Antibodies doi: 10.3390/antib15050088

Authors: Luca Guarino Giuseppe Rizzuto Vincenzo Coppolelli Alessia Scipione Mirella Scattone Luca Gargano Pietro Scribani Rossi Ester Del Duca Annunziata Dattola Giovanni Pellacani Steven Nisticò Camilla Chello

Background/Objectives: Chronic spontaneous urticaria (CSU) is a mast-cell-driven disease with autoimmune mechanisms and an incompletely clarified aetiology, in which a substantial proportion of patients remain symptomatic despite second-generation H1-antihistamines; until recently, omalizumab was effectively the only targeted option, leaving a considerable non-responder fraction. Between February 2024 and September 2025 the landscape changed: dupilumab (anti-IL-4Rα) and the first oral Bruton tyrosine kinase (BTK) inhibitor, remibrutinib, were approved for CSU; omalizumab biosimilars reached the market; the higher-affinity anti-IgE antibody ligelizumab was discontinued; and the investigational anti-KIT antibody barzolvolimab advanced to phase 3—creating new therapeutic opportunities for non-responders while leaving the choice among these second-line options unresolved by current guidelines. We provide a critical synthesis and propose an evidence-graded process for agent selection, together with a proposed repositioning of the available agents, taking the 2022 EAACI/GA2LEN/EuroGuiDerm/APAAACI guideline as the acknowledged reference. Methods: Critical narrative review with a structured but non-systematic search (PubMed, ClinicalTrials.gov) and a pre-specified evidence hierarchy (guideline to expert opinion); each clinical claim is annotated by its level of evidence. Results: Four agents are appraised in turn—omalizumab (anti-IgE, first-line), dupilumab (anti-IL-4Rα), remibrutinib (oral BTK inhibitor, small-molecule comparator) and barzolvolimab (anti-KIT, investigational). Current evidence appears to support omalizumab as first-line, with mechanism-guided second-line choices conditioned on endotype, comorbidity, prior biologic exposure and safety. Conclusions: The complexity of CSU and the incomplete response to currently available therapies support a patient-tailored approach to treatment selection.

Antibodies doi: 10.3390/antib15050087

Authors: Swati Gupta Sandip Rath Surathi Maiti Tapas Das Farhat Afrin

Cancer progression and therapeutic resistance are shaped by reciprocal dynamic interactions between malignant cells and the metabolically altered tumor microenvironment (TME). Tumor-associated hypoxia, glucose and amino acid competition, extracellular acidity, lactate accumulation, adenosine, prostaglandin E2 (PGE2) and other metabolic signals can remodel immune cell function and determine whether inflammation is tumoricidal or tumor-supportive. Although B lymphocytes have traditionally been viewed as antibody-producing cells, tumor-infiltrating B (TIL-B) cells comprise functionally heterogeneous populations that can act as antigen-presenting cells, cytokine and chemokine producers, antibody-secreting cells, cytotoxic effectors, regulatory B cells (Bregs) and organizers of tertiary lymphoid structures (TLSs). Their functional state is strongly influenced by the metabolic and spatial context in which they reside. This review focuses on the intersection of B cell biology and tumor immunometabolism, emphasizing how hypoxia, lactate, nutrient limitation, adenosine, PGE2, kynurenine and B cell-derived γ-aminobutyric acid (GABA) may shape B cell states and their interactions with myeloid and lymphoid cells. We discuss how metabolically conditioned Bregs and immunoglobulin (Ig)A-skewed humoral responses can contribute to immune suppression, whereas metabolically competent antigen-presenting, IgG-biased and TLS-associated B cell responses may support effective anti-tumor immunity. Importantly, the effects are tumor type- and context-dependent: B cell/TLS signatures are associated with favorable outcomes in several breast, lung and other solid tumors, whereas B cell-centered immune landscapes can be suppressed or neutral in pancreatic cancer and IgA-dominated responses may be unfavorable in selected malignancies. We further examine how these states may influence sensitivity or resistance to immune checkpoint blockade, chemotherapy, radiotherapy and cellular therapies. Finally, we highlight B cell metabolic pathways as potential therapeutic entry points and identify priorities for spatial metabolomics and prospective interventional studies.

Antibodies doi: 10.3390/antib15050086

Authors: Eric Ka-Wai Hui Ruben J. Boado

The blood-brain barrier (BBB) is permeable only to small lipophilic molecules, whereas hydrophilic nutrients, such as glucose and amino acids, cross the BBB via carrier-mediated transport systems. In contrast, circulating proteins do not cross the BBB. However, the BBB expresses receptors able to induce receptor-mediated transcytosis (RMT) of selective proteins across the BBB, as in the case of insulin, transferrin, and insulin-like growth factor 1 (IGF1). This led to the hypothesis that the transport of protein-based biologics to the brain may be possible by targeting BBB receptors that induce RMT. Monoclonal antibodies targeting these receptors penetrate the BBB via RMT and are distributed throughout the brain, acting as molecular Trojan horses and/or shuttle systems. A generation of brain-penetrating fusion proteins was produced targeting BBB insulin, transferrin, and IGF1 receptors, respectively, with full antibodies and/or antibody fragments acting as transport domains. These antibody-based fusion proteins were validated in various experimental models, including lysosomal storage disorders (LSDs), stroke, Parkinson’s disease, and Alzheimer’s disease (AD), respectively. Clinical trials with brain-penetrating antibody-based biologicals targeting BBB transferrin and insulin receptors have been completed or are in progress in LSDs and in AD. The aim of this review is to examine the progress made in antibody-based biologicals for CNS disorders from genetic engineering to clinical trials.

Antibodies doi: 10.3390/antib15050085

Authors: Carolina Mazeda Eduardo Dourado Raquel Freitas Patrícia Martins Liliana Saraiva Tânia Santiago Francisca Guimarães Emanuel Costa Diogo Esperança Almeida Sara Dinis Ana Sofia Pinto Alexandra Daniel Inês Genrinho Maura Couto Marília Rodrigues Maria João Salvador Ana Catarina Duarte Ana Cordeiro Maria José Santos João Eurico Fonseca Catarina Resende Inês Cordeiro

Background: Autoantibodies are central to the diagnosis and risk stratification of systemic sclerosis (SSc), but their prevalence and clinical associations may vary across populations. This study aimed to evaluate the prevalence and immuno-clinical associations of different autoantibodies in the Rheumatic Diseases Portuguese Register systemic sclerosis cohort. Methods: This was a multicentre observational registry-based study that used data from the Reuma.pt SSc module. Patients were divided according to autoantibody status, and associations between autoantibody expression and clinical data were assessed using appropriate statistical tests, with Bonferroni correction applied for multiple comparisons. Multivariable binary logistic regression was performed for clinically relevant outcomes, with adjustment for sex, age at diagnosis and disease duration. Results: A total of 1080 patients were included, 87.5% female, with a mean age at last evaluation of 60.2 ± 14.6 years and a mean disease duration of 12.4 ± 10.0 years. Limited cutaneous SSc was the most frequent clinical category (57.4%), followed by diffuse cutaneous SSc (17.7%), very early diagnosis of systemic sclerosis (VEDOSS; 12.3%), overlap syndromes (9.8%) and SSc sine scleroderma (2.8%). Antinuclear antibodies were present in 93.4% of patients. Anti-centromere antibodies (ACAs) were the most frequent SSc-specific autoantibodies (54.6%), followed by topoisomerase I antibodies (ATAs; 21.8%). ACA positivity was associated with limited cutaneous disease; older age at diagnosis; and lower frequency of interstitial lung disease (ILD), myositis and flexion contractures. ATA positivity was associated with diffuse cutaneous disease, male sex, higher modified Rodnan skin score (mRSS), digital ulcers, flexion contractures, oesophageal involvement and ILD. Among less frequent autoantibodies, anti-Pm/Scl antibodies were associated with myositis, joint involvement, calcinosis and ILD; anti-U1RNP antibodies with younger age, MCTD overlap, myositis and joint involvement; anti-RNA polymerase III antibodies (ARAs) with scleroderma renal crisis and higher mRSS; anti-U3RNP antibodies with diffuse cutaneous disease and renal involvement; and anti-Ku antibodies with overlap syndromes. Conclusions: In this large real-world SSc cohort, autoantibody status was strongly associated with distinct clinical phenotypes, confirming its value for disease stratification. While most established immuno-clinical associations were reproduced, some differences from international cohorts were observed, supporting the need for population-specific validation of autoantibody associations in SSc.

Antibodies doi: 10.3390/antib15050084

Authors: Milena Czosnek Agata Sowa Łucja Rolek Ewelina Grywalska Sebastian Mertowski Paulina Mertowska

Immune checkpoints (ICPs) are essential regulators of immune homeostasis, maintaining the balance between effective immune responses and tolerance to self-antigens. Dysregulation of ICP signaling may contribute to impaired immune surveillance, immune evasion, chronic inflammation, autoimmunity, persistent infections, and tumor progression. Consequently, ICP molecules are increasingly recognized not only as therapeutic targets but also as potential diagnostic, prognostic, predictive, and treatment-monitoring biomarkers. This review provides a comprehensive overview of the biological functions, signaling mechanisms, and clinical significance of major co-inhibitory and co-stimulatory ICP pathways, including PD-1/PD-L1/PD-L2, CTLA-4/CD28/CD80/CD86, LAG-3, TIM-3, TIGIT, BTLA, VISTA, ICOS, OX40, 4-1BB, GITR, CD27, CD40, and CD2, together with their corresponding ligands. Particular emphasis is placed on their biomarker potential in cancer and immune-mediated diseases. In addition, the review presents a bioinformatic characterization of ICP receptors and ligands based primarily on data available in UniProtKB and complementary bioinformatic resources. The analysis includes protein sequence length, molecular weight, theoretical isoelectric point, amino acid composition, subcellular localization, conserved and functional domains, protein family classification, post-translational modifications, isoforms, and selected structural features. Collectively, the available evidence indicates that ICPs constitute a structurally and functionally diverse group of immunoregulatory molecules with substantial biomarker potential. Integrating their molecular, structural, functional, and bioinformatic characteristics may improve disease classification, prognosis, patient stratification, treatment selection, and therapeutic monitoring. Such an integrated approach may also support the identification of novel biomarkers and therapeutic targets and contribute to the further development of precision and personalized medicine.

Antibodies doi: 10.3390/antib15050083

Authors: Yukari Ogura Hiroyuki Suzuki Mika K. Kaneko Yukinari Kato

Background/Objectives: Cadherin-16 (CDH16, Ksp-cadherin) possesses unique seven extracellular cadherin repeats, and its expression is restricted to normal kidney epithelium. CDH16 is downregulated in renal cell carcinoma (RCC) and is associated with poor prognosis. Therefore, developing mAbs that specifically recognize cell-surface CDH16 is essential for tumor diagnosis and for isolating CDH16-positive renal epithelial cells. Methods: Anti-human CDH16 mAbs (designated as Ca16Mabs) were developed by immunizing mice with CDH16-overexpressed tumor cells, followed by a high-throughput flow cytometry-based screening. Results: Among the 58 established Ca16Mabs, a clone, Ca16Mab-56 (IgG1, κ), specifically recognized CDH16-overexpressed Chinese hamster ovary-K1 (CHO/CDH16) cells with no detectable cross-reactivity to 21 other CDHs in flow cytometry. Ca16Mab-56 also detected endogenous CDH16 in human RCC cell lines (OS-RC-2 and KMRC-20) and normal kidney epithelial cell lines. The dissociation constant (KD) values of Ca16Mab-56 for CHO/CDH16 and OS-RC-2 were determined as 6.5 × 10−9 M and 1.2 × 10−9 M, respectively. Furthermore, Ca16Mab-56 detected endogenous CDH16 by Western blotting and showed potent staining in normal kidney tubular epithelium and clear membranous staining in renal cell carcinoma in immunohistochemistry. Conclusions: Ca16Mab-56 is a versatile tool for detecting CDH16 and has potential for tumor diagnosis.

Antibodies doi: 10.3390/antib15050082

Authors: Mikhail P. Kostinov Ivan S. Samolygo Aristitsa M. Kostinova Natalya L. Starikova Marina A. Manina Valentina B. Polishchuk Albina S. Pestova Pavel I. Zhuravlev Anton S. Antishin Svetlana I. Erdes

Background: Measles, mumps, and rubella outbreaks continue to occur despite high vaccination coverage, raising concerns about long-term vaccine-induced immunity. This study assessed serological protection against measles, mumps, rubella, and varicella in fully vaccinated Russian adolescents to identify immunity gaps and inform revaccination strategies. Methods: Serum IgG was quantified by commercial ELISA. Analytical cut-offs were: measles ≥ 0.18 IU/mL, rubella ≥ 30 IU/mL, mumps index ≥ 1.0 (index = OD/0.467), and varicella ≥ 1.0 IU/mL; equivocal results were classified as negative in the primary analysis and as positive in a sensitivity analysis. Geometric mean concentrations (GMC) were computed on the natural-logarithm scale. Results: Seropositivity differed markedly between infections (χ2 55.1; p < 0.001): rubella 81.4% (95% CI 74.6–86.7), varicella 57.7% (95% CI 49.8–65.2), mumps 53.2% (95% CI 45.4–60.9) and measles 41.0% (95% CI 33.6–48.9). GMC was lowest for measles (0.132; 95% CI 0.111–0.158) and highest for rubella (61.9; 95% CI 55.3–69.2). The measles estimate was sensitive to the treatment of equivocal results, rising to 53.2% (95% CI 45.4–60.9) at the manufacturer’s cut-off of ≥0.12 IU/mL; rubella rose to 94.9% at ≥16 IU/mL. Conclusions: Lower seropositivity rates for measles and mumps were identified among fully vaccinated adolescents, which fall below standard epidemiological benchmarks. In contrast, rubella seropositivity was robust at 81.4%. Because the measles estimate depended strongly on the assay cut-off applied, and because enzyme immunoassays are known to underestimate seroprevalence relative to neutralization testing, these findings describe the distribution of circulating antibody rather than the prevalence of susceptibility and warrant confirmation in larger representative samples with neutralization testing.

Antibodies doi: 10.3390/antib15050081

Authors: Ida Szataniak Kacper Packi

Background/Objectives: Artificial intelligence (AI) has transformed computational antibody engineering by enabling accurate prediction of antibody structures, rational optimization of therapeutic properties, and de novo antibody design. Recent advances in deep learning, protein language models, and generative AI have fundamentally changed the way antibodies are discovered and engineered. This review aims to present the historical evolution of computational antibody engineering, from early structure-based design strategies to modern AI-driven approaches, while highlighting the major computational tools, publicly available databases, current limitations, and future directions of the field. Methods: A comprehensive narrative review of the literature was conducted using PubMed, Scopus, Web of Science, and Google Scholar. Original research articles, methodological studies, and review papers published between 1985 and 2026 were evaluated. Publications were selected according to their scientific relevance, methodological quality, and contribution to the historical development of computational antibody engineering. Results: The review describes the progression of antibody engineering from phage display and structure-based computational methods to machine learning, deep learning, protein language models, and generative artificial intelligence. It summarizes key public databases supporting antibody research, discusses advances in antibody structure prediction and developability assessment, and reviews recent generative models capable of designing antibody sequences and structures. Current challenges, including limited experimental validation, dataset bias, prediction of highly flexible regions, model interpretability, and clinical translation, are also discussed. Conclusions: Artificial intelligence has fundamentally reshaped computational antibody engineering by integrating sequence, structural, and functional information into increasingly accurate predictive and generative frameworks. Although important challenges remain, recent developments indicate that AI-driven approaches will play an increasingly central role in the discovery and optimization of next-generation therapeutic antibodies.

Antibodies doi: 10.3390/antib15050080

Authors: Maya G. Cappellino Sean P. Sullivan Peyton C. High Tiffani A. Blackburn Kendra S. Carmon

Bispecific antibodies (bsAbs) and bispecific antibody–drug conjugates (bsADCs) represent promising classes of emerging targeted therapeutics with the potential to overcome tumor heterogeneity and resistance in colorectal cancer (CRC). BsAbs can simultaneously engage multiple tumor antigens or immune cells or bind two distinct epitopes within a single target, enabling mechanisms of action beyond the capabilities of monoclonal antibodies. Bispecific T cell engagers facilitate targeted destruction of tumors through immune cell recruitment, while dual immune checkpoint inhibitors enhance immune activation by blocking T cell inhibitory signals. Furthermore, bsAbs can mediate dual signaling pathway inhibition through binding multiple receptor tyrosine kinase receptors or other tumor cell surface proteins. BsADCs integrate the dual-antigen recognition of bsAbs with targeted payload delivery, utilizing receptor-mediated endocytosis to deliver potent cytotoxic payloads selectively to CRC cells, while minimizing systemic toxicity. Recent advances in bsAb engineering, linker chemistry, site-specific conjugation, and payload design have accelerated the development of bsADCs for solid tumors, including CRC. BsAbs and bsADCs provide opportunities to improve tumor selectivity, enhance internalization, overcome antigen escape, and expand the population of CRC patients eligible for targeted therapy. Emerging preclinical studies demonstrate encouraging anti-tumor activity for bispecific modalities in CRC, while early clinical trials are beginning to establish their translational potential. This review summarizes the current landscape of bsAbs and bsADCs in therapeutic development for CRC, highlighting key biological targets, engineering strategies, mechanisms of action, and clinical status. We also discuss the major challenges facing clinical translation and provide perspectives on future directions for bispecific therapies in CRC.

Antibodies doi: 10.3390/antib15050079

Authors: Yuichi Yamazaki Satoru Kakizaki Yuki Tamura Yumeo Tateyama Tatsuma Murakami Takuya Adachi Hiroki Tojima Toshio Uraoka

Background: Combination therapy with nivolumab and ipilimumab (Nivo-Ipi) has substantially improved outcomes in several advanced malignancies but is frequently associated with early-onset, severe immune-related liver injury (irAE-LI). This study aimed to identify pretreatment predictors of severe irAE-LI and to characterize its clinical course. Methods: We retrospectively analyzed 81 patients who received Nivo-Ipi combination therapy between 2018 and 2025. Severe irAE-LI was defined as liver injury requiring systemic corticosteroid therapy. Results: Severe irAE-LI developed in 11 patients (13.6%), with a median onset of 38 days after treatment initiation. More than half of the cases occurred after the first treatment cycle. This exploratory multivariable analysis identified baseline serum Krebs von den Lungen-6 (KL-6) levels of ≥400 U/mL as a potential biomarker associated with a strong preliminary risk signal for severe irAE-LI (odds ratio, 12.60; 95% confidence interval, 2.32–68.60; p = 0.003), whereas conventional liver function tests and inflammatory markers showed no significant association. The robustness of this signal was further confirmed by a sensitivity analysis excluding patients with lung adenocarcinoma (odds ratio 13.21, p = 0.005). Clinically, 54.5% of affected patients experienced steroid-refractory disease or relapse during corticosteroid tapering despite guideline-recommended high-dose corticosteroid therapy. Cytomegalovirus (CMV) reactivation was detected in 50.0% of patients tested on the basis of clinical indicators such as cytopenia or gastrointestinal symptoms, which may have contributed to a pseudo-refractory clinical course in some cases. Conclusions: Pretreatment KL-6 measurement may facilitate risk stratification before Nivo-Ipi therapy. In patients with steroid-refractory or relapsing severe irAE-LI, early introduction of mycophenolate mofetil together with CMV screening may improve clinical management.

Antibodies doi: 10.3390/antib15050078

Authors: Ponraj Prabakaran Tianlei Ying Wei Li

Antibody therapeutics now span across oncology, infectious disease, and autoimmunity, and the molecules entering development are increasingly being engineered rather than merely selected [...]

Antibodies doi: 10.3390/antib15040077

Authors: Ivan Lyagoskin Alena Agafonova Ivan Shevchenko Nina Akhtyamova-Givirovskaya Oksana Markova Marina Pantyushenko Rakhim Shukurov Ravil Khamitov

GNR-068 is a proposed biosimilar to the ustekinumab reference product (RP), which works through the antagonism of interleukin 12 and interleukin 23. Ustekinumab RP is used for the treatment of chronic inflammatory conditions, including certain forms of plaque psoriasis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis. Objectives: The purpose of the study is to study the safety, pharmacokinetics, and immunogenicity of the ustekinumab biosimilar GNR-068 and the reference drug Stelara® after a single subcutaneous administration in healthy male volunteers. Methods: This was an open-label, randomized, comparative, parallel-group clinical trial of the safety, pharmacokinetics (PK), and immunogenicity of GNR-068 (JSC GENERIUM) and Stelara® (Manufacturer: Silag AG, Switzerland; RU Holder: JOHNSON & JOHNSON) after a single subcutaneous administration of 45 mg in healthy volunteers. During the trial, 146 volunteers were screened, and 122 were randomized. Results: The results showed that PK similarity was established based on 90% confidence intervals (CIs) for the ratios of geometric means of the primary endpoints of area under the concentration-time curve from time 0 extrapolated to infinity (AUC0–∞) and maximum observed serum concentration (Cmax) being contained within the pre-specified margin of 80.00–125.00%. The incidence of total ADA was lower in the GNR-068 group compared with the reference product group. Adverse events were similar between treatment groups and consistent with the safety profile of the ustekinumab RP. Conclusions: These results indicate that GNR-068 and the ustekinumab RP share similar PK and safety profiles.

Antibodies doi: 10.3390/antib15040076

Authors: Wusong Luo Zhe Shao Tao Wang Kenghoe Lok Rongjing Zhang Yao Li

Background: Idiopathic pulmonary fibrosis (IPF) is closely associated with fibroblast proliferation, macrophage polarization, and the accumulation of extracellular matrix (ECM). Targeting CSF-1R can rebalance macrophages, reduce ECM deposition, and block pro-fibrotic signaling. Methods: A Fab fragment targeting CSF-1R was screened via phage display. After sequence optimization, it was constructed into an IgG1 antibody (BC006). In vitro activity, domain binding, and signaling blockade were investigated, and in vitro safety indicators were evaluated. Efficacy was assessed using an induced IPF organoid-on-a-chip and bleomycin-induced mouse model. In vivo safety evaluation was conducted in cynomolgus monkeys. Results: BC006 showed an EC50 value of 226 ± 57 nM and a KD of 30 ± 2 nM. It specifically bound to the Ig4-5 domain of CSF-1R by inhibiting receptor dimerization without blocking ligand binding. It could dose-dependently inhibit the differentiation of monocytes into M2 macrophages and exert anti-IPF effects. In the induced IPF organoid-on-a-chip model, BC006 maintained lung barrier function and decreased α-SMA and Collagen I. It also improved lung function and attenuated the degree of fibrosis in the mouse model. Moreover, BC006 had no ADCC, CDC, cytokine release, or hemagglutination, and demonstrated favorable safety profiles in cynomolgus monkeys. Conclusions: BC006 is a novel anti-fibrosis antibody specifically targeting the CSF-1R Ig4-5 domain and offers a new therapeutic strategy for IPF.

Antibodies doi: 10.3390/antib15040075

Authors: Preanka Mudaly Max Lee Aishwarya Bhatta Craig Thompson

Influenza remains a persistent global health threat, accounting for an estimated one billion infections and 350,000–600,000 deaths worldwide annually. Beyond substantial mortality and morbidity, influenza imposes major socio-economic costs through healthcare utilisation and productivity losses, amounting to tens of billions of dollars each year. Current seasonal vaccination strategies are constrained by rapid antigenic evolution, which necessitates frequent vaccine reformulation and contributes to suboptimal vaccine efficacy across seasons. These drawbacks highlight the need for a universal influenza vaccine (UIV) that protects against all seasonal and potential pandemic influenza strains via a single dose. This review evaluates the feasibility of a UIV as an alternative to a seasonal influenza vaccine (SIV) by consolidating evidence from current UIV candidates in preclinical and clinical development across a diverse range of platforms and target epitopes. Preclinical studies and clinical trials have demonstrated the safety and tolerability of multiple candidates, as well as their ability to elicit broadly cross-reactive immune responses. However, several challenges remain regarding the long-term efficacy of these vaccines. In particular, pre-existing host immunity can shape subsequent vaccine responses, with immune imprinting introducing biases toward previously encountered influenza strains, potentially reducing vaccine effectiveness. In addition, the genetic plasticity of influenza viruses, driven by ongoing mutations and genetic reassortment, raises concerns about the ability of a single vaccine formulation to maintain broad and durable protection. Overall, several UIV candidates have shown promising early results. Although ecological and immunological barriers remain, a UIV could become a viable alternative to, or eventual replacement, for SIVs.

Antibodies doi: 10.3390/antib15040073

Authors: Hyun Jin Shin Chaerin Kwon Jeeyoung Oh

Background: Ocular myasthenia gravis (OMG) can remain restricted to ocular symptoms or progress to generalized myasthenia gravis (GMG). However, the rate of generalization and its predictors may vary across populations, and data on South Korean patients remain inadequate. This study investigated the conversion rate from OMG to GMG and identified clinical, serologic, electrophysiologic, and thymic predictors of this generalization in a South Korean cohort. Methods: This retrospective cohort study included 89 patients who initially presented with OMG and were followed up for at least 2 years. The primary outcome was conversion to GMG during follow-up. The investigated predictors included demographic variables, initial ocular manifestations, acetylcholine receptor antibody (AChR-Ab) status and titer, repetitive nerve stimulation test (RNST) findings, thymic abnormalities, and immunosuppressive treatment. Kaplan–Meier analyses and Cox proportional-hazards models were used to evaluate the GMG-free survival rate and risk factors for conversion. Results: During follow-up, 10 patients (11.2%) progressed to GMG, with this conversion occurring within 2 years in 8 patients (9.0%). All converters were AChR-Ab positive. AChR-Ab positivity, RNST positivity, thymic abnormality, and thymoma were associated with GMG conversion in univariable analyses. RNST positivity in any tested muscle (hazard ratio [HR] = 12.66, 95% confidence interval [CI] = 2.68–59.76, p = 0.001) and thymoma (HR = 10.67, 95% CI = 3.06–37.27, p < 0.001) was associated with generalization. These associations persisted in the restricted multivariable Cox model. Conclusions: The rate of GMG conversion was relatively low in this South Korean OMG cohort, and most conversions occurred within 2 years. The presence of AChR-Ab positivity, RNST abnormality, or thymoma may characterize high-risk patients who require closer monitoring during the early disease course.

Antibodies doi: 10.3390/antib15040074

Authors: Orsola Crespi Umberto Santaniello Luca Cangialosi Ambra Bonvicino Michela Ortoncelli Francois Rosset Luca Mastorino Simone Ribero Pietro Quaglino

Background/Objectives: Congenital ichthyoses (CI) are inherited disorders of keratinization characterized by epidermal barrier dysfunction, abnormal desquamation, and variable degrees of inflammation. Increasing evidence suggests a role for IL-23/Th17-mediated immune dysregulation in some CI subtypes, providing a rationale for targeted biologic therapies. Secukinumab, an anti-IL-17A monoclonal antibody, has emerged as a potential therapeutic option, although real-world evidence remains limited. This study evaluated the effectiveness of secukinumab in adult patients with severe CI. Methods: We performed a retrospective monocentric case series including eight adult patients with severe congenital ichthyosis treated with secukinumab for at least six months and followed for up to 56 months. Clinical outcomes included Investigator Global Assessment (IGA) scores for erythema, scaling, scalp involvement, and palmoplantar hyperkeratosis, together with pruritus assessed by Numeric Rating Scale (NRS). Results: Erythema and scaling showed most of their improvement during the first six months of treatment, after which mean clinical scores remained relatively stable throughout follow-up. At the last available assessment, improvement rates were 100% for erythema, 87.5% for trunk and limb scaling, 100% for scalp scaling, and 75% for palmoplantar hyperkeratosis. Hyperkeratosis showed a less pronounced response than inflammatory manifestations. Pruritus improved within the first 3–4 months of treatment and was sustained over time, with mean NRS scores decreasing from 6.5 at baseline to below 4 within 3–4 months. No discontinuations due to adverse events or lack of efficacy were recorded. Conclusions: Secukinumab was associated with clinically meaningful improvements in inflammatory manifestations of CI, particularly erythema and pruritus, while showing a more limited effect on hyperkeratotic features. These findings support the potential role of IL-17 inhibition in selected CI phenotypes.

Antibodies doi: 10.3390/antib15040072

Authors: Nicole Ivaska Laura Rothschild

Background: Sex-related differences in SARS-CoV-2 viral susceptibility, severity, and recovery are prevalent. Factors such as age and biological variation provide some explanation for immune response, yet the use of biological sex as a variable may limit understanding of underlying individual mechanisms related to endocrine–immune function involved in long-term immune memory. Endogenous sex hormone concentrations provide a means to assess the magnitude or durability of antibody persistence and disease recovery. Objective: This study examined salivary concentrations of 17β-estradiol and testosterone, as well as the number of weeks since prior SARS-CoV-2 viral infection. The study aimed to determine whether endogenous sex hormones contributed additional explanatory value to antibody persistence beyond biological sex. Methods: A cross-sectional study included 75 college students (63% female) who reported weeks since previous SARS-CoV-2 infection (M = 15.41, SD = 7.13), confirmed through a positive SARS-CoV-2 anti-N rapid antibody test. Participants provided saliva samples, which were tested for concentration of 17β-estradiol and testosterone. Hierarchical multiple regression analyses examined the effects of biological sex and endogenous sex hormones on the persistence of SARS-CoV-2 antibodies following natural infection. Results: 17β-estradiol was the strongest positive predictor of SARS-CoV-2 antibody persistence (β = 0.63, p < 0.001). Departing from previous findings, biological sex was not a significant predictor of antibodies targeting the SARS-CoV-2 N protein. Conclusions: Findings underscored the role of estradiol on immune recovery in a healthy young adult population. Individual hormone variation appears more informative than sex classification for understanding antibody persistence and immune response.

Antibodies doi: 10.3390/antib15040071

Authors: Tomohiro Watanabe Masatoshi Kudo

Inflammatory bowel diseases (IBDs), chronic inflammatory disorders of the gastrointestinal tract, are classified into Crohn’s disease (CD) and ulcerative colitis (UC). Despite differing clinical manifestations, both CD and UC share a common immunopathogenesis, involving excessive production of proinflammatory cytokines by macrophages and dendritic cells. Tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), and IL-23 are prototypical proinflammatory cytokines associated with CD and UC development, and biologics targeting these cytokines are widely used for treating patients with IBD. Antibodies (Abs) against IL-12 and IL-23 decrease intestinal inflammation by binding to the soluble forms of IL-12 and IL-23, respectively. Notably, anti-TNF-α Abs induce remission by neutralizing soluble TNF-α as well as membrane-bound TNF-α (mTNF-α). The binding of anti-TNF-α Abs to mTNF-α enables the diverse functions of IBD-specific TNF-α-inhibitors. Multiple modes of action of anti-TNF-α Abs for IBD include complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity, apoptosis of cells expressing mTNF-α, apoptosis of lamina propria T cells, and induction of regulatory T cells and macrophages. In this article, we introduce the diverse actions of anti-TNF-α Abs beyond TNF-α neutralization and discuss how these unique properties of anti-TNF-α Abs contribute to the efficient downregulation of IBD-associated inflammation.

Antibodies doi: 10.3390/antib15040070

Authors: Raeshun T. Glover Robert W. Maitta

Sickle cell disease (SCD) represents one of the most complex hematological diseases leading to a lifetime of physiological crises characterized by chronic hemolysis, inflammation, hypoxia, and anemia with changes to body systems that result in patients having shorter lifespans. Transfusions, either simple or as part of red cell exchanges, are often needed to lower the hemoglobin S levels to minimize the possibility of sickling of red blood cells (RBCs) and provide patients with greater oxygen carrying capacity. However, hemolysis in SCD patients is a common finding/presentation of the disease, especially during acute crises. One of the ensuing complications of a life of transfusions is the development of alloantibodies to RBC antigens despite partial or extended matching. This is further complicated by formation of autoantibodies even in the setting of RBC matching, suggesting that a hyperactive immune response in these patients is primed to respond with formation of antibodies. In a sub-cohort of patients, no antibodies are detected despite extensive investigation, and the ensuing hemolysis requires minimizing exposure to RBC transfusions to avoid developing a greater hemolytic process. Instead, immunosuppression or monoclonals that target complement or cytokines shown to be involved in this type of hemolysis are necessary. In this context, this narrative review will present antibody-dependent and antibody-independent mechanisms of hemolysis in SCD, including therapeutic approaches that target specific areas of the immune response that are possibly involved in the destruction of RBCs.

Antibodies doi: 10.3390/antib15040069

Authors: Antonella Bruzzese Enrica Antonia Martino Santino Caserta Maria Eugenia Alvaro Nicola Amodio Eugenio Lucia Virginia Olivito Caterina Labanca Francesco Mendicino Fortunato Morabito Ernesto Vigna Massimo Gentile

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts and the persistence of leukemic stem cells (LSCs) within a profoundly remodeled bone marrow (BM) microenvironment. Despite advances in molecular stratification and the introduction of targeted agents, long-term outcomes remain unsatisfactory, particularly in older and high-risk patients. Increasing evidence indicates that leukemogenesis and treatment resistance are critically sustained by a permissive immune milieu, in which LSCs, myeloid-derived suppressor cells, leukemia-associated macrophages, and dysfunctional T and NK cells shape an immunosuppressive “leukemic niche.” This evolving understanding has renewed interest in immune-based strategies capable of restoring effective antitumor immunity. Bispecific antibodies (bsAbs) are engineered molecules designed to engage AML-associated antigens while simultaneously recruiting and activating immune effector cells, most commonly T cells or NK cells. By promoting immune synapse formation independently of major histocompatibility complex expression and conventional co-stimulatory pathways, bsAbs can overcome several mechanisms of immune escape. In this review, we summarize the biological rationale for immunotherapy in AML, with a focus on the role of the BM microenvironment and immune dysregulation. We then discuss the structural and functional properties of IgG-like and non-IgG-like bsAbs, key antigenic targets such as CD33, CD123, CD70 and others, and the main T-cell- and NK-cell-engaging platforms under clinical investigation. Finally, we highlight emerging clinical data, principal toxicities, and the challenges of integrating bsAbs into existing treatment algorithms, including combinations with hypomethylating agents, BCL-2 inhibitors, and allogeneic stem cell transplantation. A deeper understanding of AML immune biology and antigen expression patterns will be essential to optimize bsAb design, maximize therapeutic benefit, and minimize on-target off-tumor toxicity.

Antibodies doi: 10.3390/antib15040068

Authors: Arpita Deb Barbara L. F. Kaplan

Background/Objectives: Aryl hydrocarbon receptor (AHR) ligands are known to suppress antibody production. However, it remains unclear whether this suppression translates into altered antibody-mediated Fcγ receptor signaling in human cells. This study aimed to investigate the effects and mechanisms by which AHR ligands regulate antibody production and antibody-triggered responses in human peripheral blood mononuclear cells (PBMCs). Methods: PBMCs were isolated from blood obtained from anonymous healthy donors. PBMCs were stimulated with pokeweed mitogen (PWM) to induce IgG1 production or Strept-Biotin IgG1/IgG2 immune complexes to induce antibody-triggered signaling. Before any stimulation, cells were pretreated with vehicle (0.01% DMSO) or AHR ligands TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), ITE (2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester), or FICZ (6-formylindolo[3,2-b]carbazole), or the proligand I3C (indole-3-carbinol). Results: Our data revealed that PWM stimulation significantly increased IL6 and IL1B gene expression and PWM-induced IL-6 cytokine secretion, which was significantly suppressed by TCDD, ITE, FICZ, and I3C. However, only TCDD was able to suppress PWM-stimulated IgG1 antibody production. Transcriptomic analysis using I3C revealed upregulated AHR-responsive genes such as CYP1A1, CYP1B1, AHRR and TIPARP in PWM-stimulated human PBMCs. Notably, I3C downregulated genes TRAPPC9 and C1QTNF3, which play a role in NF-κB-associated inflammatory signaling. It also revealed potential sex differences in I3C-mediated gene modulation associated with B cell function, Ig expression, FcγR signaling, and inflammatory pathways. Lastly, IgG1 and IgG2 immune complex-stimulated IL-6 cytokine secretion was significantly suppressed by TCDD, whereas I3C showed modest, although not statistically significant, suppression. Conclusions: Overall, these findings demonstrate that AHR activation by TCDD suppressed antibody production and IgG-mediated immune signaling in human PBMCs.

Antibodies doi: 10.3390/antib15040067

Authors: Tatiana Bezukladnikova Svetlana Zamorina Sergey Lazarev Mikhail Rayev

Hybridoma technology remains one of the most reliable and widely used platforms for generating highly specific monoclonal antibodies for use in diagnostics, fundamental research, and clinical practice. Moreover, the combination of unlimited proliferative capacity with the preservation of a key B-lymphocyte function, antibody production, enables hybridoma cells to support a comprehensive functional evaluation of cellular responses. Thus, by integrating analysis of proliferation, viability, and productivity, hybridoma-based approaches enable the detection of differential modulatory effects and offer a nuanced assessment of compound bioactivity. This review provides a comprehensive analysis of the functional parameters of hybridoma cells: viability, proliferation, and productivity, as well as the methods used for their evaluation. The main stages of hybridoma cell generation, advances in hybridoma technology, and current applications of hybridoma cells are also reviewed. A key aspect of this review is the differential modulation of functional parameters of hybridoma cells. Modulation of culture conditions and bioactive compounds can differentially influence growth dynamics and specific antibody yield, often revealing an inverse relationship between proliferation and productivity.

Antibodies doi: 10.3390/antib15040066

Authors: Felix Meiser Julia Harwardt Christoph Hahn Marta Lustig Thomas Valerius Harald Kolmar

Background/Objectives: CD89 (FcaRI), the receptor for immunoglobulin A (IgA), is expressed on myeloid cells, including neutrophils, monocytes, and macrophages. It is known to mediate potent effector functions against tumor cells, but the clinical development of IgA-based therapeutics is hindered by manufacturing challenges and IgA’s short plasma half-life. Methods: Using yeast display, we generated EGFR × CD89 bispecific common light chain antibodies and investigated their biophysical properties, neutrophil-mediated cytotoxicity and macrophage phagocytosis in vitro. Results: CD89-targeting constructs induced potent neutrophil cytotoxicity and macrophage phagocytosis in vitro. A symmetric 2 + 2 IgG1 Fc-silenced variant showed the most consistent potency across neutrophil and macrophage effector functions. Fc silencing enhanced neutrophil ADCC and did not induce detectable neutrophil or PBMC fratricide under the conditions tested, suggesting a limited propensity for off-target immune cell killing. Conclusions: These findings support further preclinical evaluation of CD89-targeting bispecific antibodies and indicate that Fc silencing may differentially shape neutrophil- and macrophage-mediated antitumor activity.

Antibodies doi: 10.3390/antib15040065

Authors: Ioannis Ogrotis Konstantinos Drouzas Evangelia Pantzopoulou Petros Nikolopoulos Ioannis Kotsantis Amanda Psyrri George Liapis Sophia Lionaki

Background/Objectives: Acute interstitial nephritis (AIN) is a cause of acute kidney injury (AKI) during immune checkpoint inhibitor (ICI) therapy, but biopsy-confirmed data are limited. We estimated institutional proportions of biopsy-confirmed AIN clinically attributed to ICI exposure (ICI-AIN) and characterized its presentation, pathology, treatment, and renal outcomes. Methods: We retrospectively reviewed native renal biopsies performed at Attikon University Hospital from February 2021 through December 2025. Cases were included when ICI exposure preceded AKI and the treating nephrology team documented clinicopathologic attribution to ICI exposure. Results: AIN was identified in 15 of 339 native renal biopsies; 12 cases were attributed to ICI exposure, representing 3.5% of native renal biopsies and 0.8% of 1472 unique ICI-treated patients. Median serum creatinine increased from 1.05 mg/dL at baseline to 3.50 mg/dL at biopsy assessment. During the AKI episode, 10 patients (83.3%) met Kidney Disease: Improving Global Outcomes (KDIGO) criteria for stage 3 AKI. All patients had pyuria, negative urine cultures, and subnephrotic proteinuria. Hematuria and peripheral eosinophilia occurred in four and two patients, respectively. All patients received corticosteroids, and none required kidney replacement therapy. Complete, partial, and absent recovery occurred in eight, two, and two patients, respectively; under the stricter baseline-relative definition, the corresponding numbers were five, five, and two. Conclusions: Biopsy-confirmed ICI-AIN was infrequently detected. Severe AKI was common, urinary findings were nonspecific, and residual renal dysfunction frequently persisted after corticosteroid treatment.

Antibodies doi: 10.3390/antib15040064

Authors: Victor Ovchinnikov Martin Karplus

Background: The elicitation of broadly neutralizing antibodies against conserved antigenic epitopes has been a focus of universal vaccine research. To facilitate immunofocusing on the conserved influenza hemagglutinin (HA) stalk, we designed headless trimeric antigens, initially focusing on subtypes H1, H3, and H5, and subsequently expanding to eight additional HA subtypes. Results: The designs were first evaluated in silico; they were predicted to fold correctly by AlphaFold2, and remained stable in molecular dynamics simulations in isolation, as well as bound to a broadly neutralizing antibody. The antigens expressed in HEK293-derived cells in high yields, and eluted predominantly as trimers in SEC-HPLC. Future work will explore the use of mosaic nanoparticles decorated with headless antigens of various subtypes for the optimal elicitation of broadly neutralizing anti-influenza antibodies. Overall, our study advances the use of headless HA trimers as modular antigens toward a universal influenza vaccine.

Antibodies doi: 10.3390/antib15040063

Authors: Michał Kołodziej Dorota Antos

Although mAbs have great therapeutic potential, their use in medicine is currently limited by the high cost of their manufacturing. Significant developments in upstream processing technologies have caused downstream processing (DSP) to become the manufacturing cost-driver. DSP consists of a number of operations included in the capture, polishing, and formulation steps that contribute to excessive material and buffer consumption. This is particularly true for the capture and polishing steps, in which tedious and costly chromatographic operations are involved to ensure an adequate purity level of the medical product. The final formulation step also increases the burden of buffer consumption. This review focuses on those time- and material-consuming DSP operations and describes key issues and challenges related to their realization. In each of the steps, capture, polishing, and formulation, the platform processing approaches are presented as well as directions for their development. In addition, we present alternative nonchromatographic approaches that can potentially be used in the capture and polishing steps, such as precipitation or extraction. Furthermore, we describe mAb processing by crystallization, which can potentially serve as an alternative platform in both polishing and formulation steps.

Antibodies doi: 10.3390/antib15040062

Authors: Ginka Cholakova Alexandra Atanasova Alexandra Kapogianni Dobroslav Kyurkchiev Bogdan Penev Ivanka Tsacheva

Background/Objectives: The autoimmune disorder Systemic Lupus Erythematosus (SLE) is characterized by increased titers of autoantibodies with different specificities against autoantigens, including the complement proteins C1q, C3 and Factor H. SLE is characterized by chronic inflammation and tissue damage due to the secretion of pro-inflammatory molecules and a tissue deposition of immune complexes formed by autoantibodies and their target antigens. The inflammatory process in SLE is maintained by the phospholipase A2 (PLA2) enzymes generating pro-inflammatory lipid mediators. Hereditary and environmental factors trigger SLE, with increased genetic heritability in first-degree relatives of SLE patients. Methods: A cohort of 48 healthy FDRs of SLE patients was analyzed with the ELISA method for the presence of antibodies to complement proteins C1q, C3 and Factor H, with a focus on detecting autoepitopes both on immobilized and soluble C1q and its globular fragments ghA, ghB and ghC. The total serum PLA2 activity of FDRs was measured using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid (NOBA). Results: Only C1q, and specifically its globular domains in both an immobilized and soluble state, was targeted by antibodies in the healthy FDRs similarly to the pattern established in SLE patients. In contrast, C3 and Factor H which are known autoantigens in SLE were not found as targets for the antibodies in the analyzed FDRs. Some of the FDRs showed increased serum PLA2 activity, which correlated weakly with anti-C1q antibodies. Conclusions: C1q and its globular domains are estimated as autoantigenic molecules for binding in the analyzed FDRs of SLE patients.

Antibodies doi: 10.3390/antib15040061

Authors: Bogdan Ghilencea Bianca Ilinca Moroianu Ancuța-Iuliana Năstac Ioana-Stefania Bostan Anca Panaitescu Claudia Mehedințu Nicolae Gică

Human Papillomavirus (HPV) infection is the leading cause of cervical cancer, presenting a significant global health challenge. While natural infection is widespread, the resulting immune response is often characterized by weak, delayed, and type-specific antibody production, offering unreliable protection against reinfection. This review provides a comparative analysis of natural versus vaccine-induced immunity, focusing on antibody kinetics, duration of protection, and cancer prevention efficacy. A comprehensive search of the literature from the last decade was conducted using PubMed, ScienceDirect, and Web of Science. The findings demonstrate that, unlike natural immunity, which is dominated by cellular responses with often incomplete seroconversion, prophylactic vaccination induces high titers of neutralizing IgG antibodies against the L1 capsid protein. These responses are durable, with protection persisting for over a decade, and recent data support the high efficacy of single-dose regimens. Furthermore, vaccination has shown utility in reducing infection persistence in HPV-positive individuals and provides critical protection in immunocompromised groups. Consequently, vaccine-induced immunity is consistently superior to naturally acquired immunity, supporting World Health Organization recommendations for universal vaccination as the primary intervention for reducing the global burden of HPV-related malignancies.

Antibodies doi: 10.3390/antib15040060

Authors: Christian D’Elia Giada Santagata Serena Guiducci Holger Bang Mariangela Manfredi Maria Infantino Maurizio Benucci

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is one of the most severe extra-articular manifestations of rheumatoid arthritis (RA), requiring reliable biomarkers for early detection. This scoping review synthesized current evidence regarding the diagnostic performance and clinical associations of anti-mutated citrullinated vimentin (anti-MCV) antibodies in patients with RA-ILD. A comprehensive literature search was conducted across PubMed/MEDLINE, Embase, Scopus, and the Cochrane Library. Following systematic screening, two observational studies met the predefined inclusion criteria. Both included studies reported significantly higher anti-MCV positivity rates and/or serum levels in patients with RA-ILD compared with RA patients without pulmonary involvement. Specifically, one study identified an independent association between anti-MCV positivity and RA-ILD, while the other demonstrated significant correlations between anti-MCV titers and pulmonary function impairment, as well as disease activity markers. However, substantial heterogeneity was observed across the studies regarding assay platforms, positivity thresholds, and diagnostic cut-offs, which limits the direct comparability of results. While anti-MCV antibodies represent promising candidate biomarkers for RA-ILD, current evidence remains limited and is insufficient to establish definitive diagnostic, prognostic, or pathogenic significance. Consequently, larger, prospective, and multi-center studies utilizing standardized anti-MCV assay protocols are necessary to rigorously evaluate the clinical utility of these antibodies in the management of RA-ILD.

Antibodies doi: 10.3390/antib15040059

Authors: Reina Ito Hiroyuki Suzuki Kenichiro Ishikawa Kazutake Yagi Akira Ohkoshi Yukio Katori Mika K. Kaneko Yukinari Kato

Background/Objectives: Cadherin-17 (CDH17, LI-cadherin) is a non-classical cadherin with an atypical structure and unique functions. CDH17 expression is restricted to normal intestinal epithelium. Furthermore, CDH17 functions as an oncoprotein that promotes tumor migration and invasion in colorectal, gastric, and pancreatic cancers. Therefore, CDH17 is an important diagnostic marker and therapeutic target. The CDH17-directed strategies, including monoclonal antibodies (mAbs), bispecific Abs, antibody–drug conjugates (ADCs), and chimeric antigen receptor (CAR) T cells, have been evaluated in preclinical and clinical studies. Therefore, developing mAbs that specifically recognize cell surface-expressing CDH17 is essential for advancing both tumor diagnosis and therapy. Methods: Anti-human CDH17 mAbs (named Ca17Mabs) were developed by immunizing a mouse with CDH17-overexpressed cells and a high-throughput screening using flow cytometry. Results: Among Ca17Mabs, a clone, Ca17Mab-5 (IgG1, κ) specifically recognized CDH17-overexpressed Chinese hamster ovary-K1 (CHO/CDH17) cells with no detectable cross-reactivity to 21 other CDHs by flow cytometry. Ca17Mab-5 also detected endogenous CDH17 in human colorectal cancer cell lines, COLO201 and COLO205. The apparent dissociation constant (KD) values of Ca17Mab-5 for CHO/CDH17 and COLO205 were estimated as 1.5 × 10−8 M and 1.3 × 10−8 M, respectively. Furthermore, Ca17Mab-5 detected endogenous CDH17 by Western blotting. In immunohistochemistry, Ca17Mab-5 exhibited clear membranous staining in normal colon epithelium, colorectal, gastric, and pancreatic cancers. Conclusions: Ca17Mab-5 is a versatile tool for detecting CDH17 and has potential for tumor diagnosis.

Antibodies doi: 10.3390/antib15040058

Authors: Hanna Cholerzyńska Gabriela Kot Łukasz Świątek Bogna Grygiel-Górniak

Background: Antinuclear antibodies (ANA) can be detected in patients with rheumatoid arthritis (RA) and pose many diagnostic challenges, especially when RA presents an atypical course and requires differentiation from other systemic connective tissue diseases (sCTDs). This study assessed ANA fluorescence patterns and immunoblot profiles, as well as the relationships between ANA titers, antibody expression intensity, and markers of disease activity in patients with RA. Methods: This single-center, cross-sectional, observational study included 81 RA patients (53 ANA-positive) meeting the 2010 ACR/EULAR classification criteria. ANA titers and fluorescence patterns were assessed using indirect immunofluorescence. Anti-extractable nuclear antigen (ENA) autoantibody profiles and expression intensity were assessed using immunoblot analysis. Demographic, clinical, and laboratory data were obtained. Spearman’s rank correlation coefficient was used to analyze the relationship between ANA titers and selected variables. Univariate and multivariate logistic regression analyses were performed to identify factors associated with ANA positivity. Results: The cohort consisted primarily of women (86.4%) with moderate disease activity. ANA fluorescence patterns were heterogeneous, with nucleolar and homogeneous patterns most frequently observed. Immunoblot analysis also revealed diverse autoantibody profiles without a clearly dominant specificity. Ro-52, SS-A, and Sm antibodies were detected more frequently, although their prevalence remained relatively low. No statistically significant correlations were found between ANA titers and inflammatory markers, serological parameters, or disease activity indices. Conclusions: RA patients with positive ANA demonstrated marked immunological heterogeneity, without concomitant symptoms of sCTD. A positive result in RA may reflect generalized immune dysregulation rather than a distinct clinical subtype. Further studies with larger cohorts are needed to clarify the clinical significance of ANAs in rheumatoid arthritis.

Antibodies doi: 10.3390/antib15040057

Authors: Marco Allinovi Luca Malatesta Tiziana Biagioli Elisabetta Antonioli Federico Perfetto

Daratumumab, a human IgG1 monoclonal antibody targeting CD38, is widely used in multiple myeloma and AL amyloidosis. Despite its clinical success, many patients fail to achieve durable responses or relapse, underscoring the importance of understanding resistance mechanisms. Drawing on experience from other better-studied monoclonal antibodies, resistance to daratumumab can be categorized into four main mechanisms: (1) reduced CD38 expression on plasma cells; (2) increased expression of complement inhibitory proteins (CD55/CD59), impairing complement-mediated cytotoxicity; (3) reduced drug bioavailability due to urinary loss in non-selective nephrotic syndrome; and (4) the development of neutralizing anti-daratumumab antibodies. Anti-drug antibodies (ADAs) may represent a potential mechanism of treatment failure through effects on pharmacokinetics, efficacy, and safety, even in patients on daratumumab therapy. Seven different trials have tested anti-daratumumab antibodies. Among them, anti-daratumumab antibodies were identified in only 0–2.4% of patients, and only in a small portion of these has it been proven to be neutralizing. Overall, ADAs appear rare, but these findings are likely underestimated due to short follow-up and suboptimal timing of assessment. In conclusion, standardized ADA monitoring, particularly months after treatment interruption or in cases of inadequate response or infusion-related reactions, may improve patient management and therapeutic outcomes.

Antibodies doi: 10.3390/antib15040056

Authors: Eva Agustriana Koki Murozono Kosuke Minamihata Riko Nishioka Noriho Kamiya

Background/Objectives: The use of cross-linking enzymes for site-selective and efficient antibody modification has attracted considerable attention. Microbial transglutaminase (MTG)-mediated labeling of IgG at Gln295 has emerged as a promising strategy for preparing antibody–drug conjugates (ADCs). By contrast, selective modification of a specific Lys residue on native antibody surfaces using MTG remains challenging because most Lys residues exhibit low intrinsic reactivity. Here, we address this challenge by exploiting enzyme–antibody proximity together with screening for highly reactive Gln-donor substrates from a random peptide library. Methods: Reactive Gln-donor peptide substrates were first identified from a seven-amino-acid phage-displayed peptide library using a reactive Lys-containing peptide as bait. Based on the obtained sequence, an azide-functionalized Gln-donor peptide suitable for click chemistry was designed. Results: The designed substrate enabled efficient Lys65-selective modification of Fab fragments using a fusion of an engineered MTG zymogen and protein G (EzMTG-pG), followed by functionalization through click chemistry to yield fluorescent Fab conjugates. Conclusions: These results provide practical guidelines for substrate design in MTG-mediated site-selective protein modification.

Antibodies doi: 10.3390/antib15040055

Authors: Anjali Shenoy Daniel J. Falconer Adam W. Barb

Background: The interaction between human immunoglobulin G (IgG)1 Fc and the Fc gamma receptor (FcγR) IIIa/CD16a elicits protective immune responses. Antibody N-glycosylation stabilizes the FcγR-binding interface and is thus essential for interaction with wildtype IgG1 Fc. Furthermore, the N-glycan introduces substantial compositional and functional heterogeneity, with distinct glycoforms providing different affinities and discrete responses in vivo. Accordingly, various engineering endeavors to improve antibody binding strive to boost the therapeutic efficacy of monoclonal antibodies but do not directly address compositional heterogeneity. Objective: Here, we describe a previously unexplored approach to engineer IgG1 Fc. We eliminated carbohydrate heterogeneity by removing the N-glycan but stabilizing the FcγR-binding interface with disulfide bonds. Conclusions: These newly generated Fc domains served as a starting point for protein engineering through yeast surface display to enhance receptor-binding affinity. We recovered Fc variants from this approach that demonstrated FcγRIIIa binding affinities comparable to the starting sequence and thus serve as a proof-of-principle for this strategy.

Antibodies doi: 10.3390/antib15040054

Authors: Elyse Moritz Renato Cerqueira Juliana Oliveira Martins José O. Bordin

Immune-mediated neutropenias comprise a heterogeneous group of disorders characterized by antibody-mediated destruction of neutrophils, in which the detection of anti-neutrophil antibodies remains a significant diagnostic challenge. Human neutrophil antigens (HNAs) are key targets in both autoimmune and alloimmune conditions, and their identification requires an integrated laboratory approach combining serological assays, HNA genotyping, and clinical evaluation. However, variability in assay sensitivity, the presence of low-titer or conformationally dependent antibodies, and interference from anti-HLA antibodies may lead to inconclusive or misleading results. This review summarizes the immunological mechanisms underlying anti-HNA antibody-mediated neutropenia and critically evaluates current laboratory methods, including cell-based and bead-based assays. The role of HNA genotyping in supporting antibody identification and improving diagnostic accuracy is also discussed. In addition, we highlight the importance of interpreting serological findings according to antibody specificity and clinical context. An integrated and multidisciplinary diagnostic approach is essential to ensure accurate diagnosis and appropriate clinical management, while emerging technologies may further improve antibody detection in the future.

Antibodies doi: 10.3390/antib15040053

Authors: Aidong Qiu Ruiyuan Wang Yangyingjie Bai Bowen Zhang Xinyu He Jiani Xie Jianghai Liu

Background: The mucosal barrier presents a significant challenge for non-invasive delivery of macromolecular therapeutics, often requiring administration with poor bioavailability and increased toxicity risks. The polymeric immunoglobulin receptor (pIgR) contains an extracellular secretory component (SC) for immunoglobulin binding and a membrane-anchored stem domain capable of apical-to-basolateral transcytosis. We hypothesized that targeting the stem domain could enable active drug transport across mucosal barriers. Methods: Using phage display, we identified four high-affinity nanobodies against human and murine pIgR. Two lead candidates (3LTHMP-4 and 3LTHMP-5) demonstrated efficient apical-to-basolateral transport in vitro (Transwell assays) and in vivo (fluorescence imaging). Engineered bispecific antibodies fusing these nanobodies with anti-IL-5 mAb reslizumab were administered via inhalation in a murine asthma model at one-tenth the intraperitoneal reslizumab dose. Resluts: The bispecific antibodies showed significant therapeutic efficacy, while reslizumab alone at equivalent concentrations failed to demonstrate efficacy. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) revealed that both 3LTHMP-4 and 3LTHMP-5 specifically bind to the pIgR stem domain (residues 578–612), a region distinct from the dimeric IgA binding site. Conclusions: These findings suggest that stem domain-specific binding may facilitate transport across the mucosal barrier while preserving native receptor physiology, offering a potential strategy for effective transmucosal delivery of biologics.

Antibodies doi: 10.3390/antib15040052

Authors: Junfang Hao Qian Xu Yanyan Cui Wenlong Wang Kai Huang

Background: Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus–host interactions. Methods: Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Results: Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of −83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (KD = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). Conclusions: This study not only provides novel candidate molecules for elucidating the mechanism of HS–virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs.

Antibodies doi: 10.3390/antib15030051

Authors: Polona Žigon Katja Lakota Katarina Ogrinc Petra Bogovič Franc Strle

Objectives: Borrelia burgdorferi sensu lato, a spirochete bacterium responsible for Lyme borreliosis—the most common tick-borne infection in North America and Europe—can trigger the production of antiphospholipid antibodies. These antibodies target host lipids such as cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS), which the spirochete incorporates into its membrane from the surrounding environment. Although antiphospholipid antibodies are typically associated with antiphospholipid syndrome (APS), they may also arise during infections, including Lyme borreliosis. This study aimed to develop and optimize several enzyme-linked immunosorbent assays (ELISAs) for measuring various antiphospholipid antibodies in patients with Lyme borreliosis. Methods: Thirty patients diagnosed with Lyme borreliosis were enrolled: ten with solitary erythema migrans (EM), ten with multiple EM (MEM), and ten with late manifestations known as acrodermatitis chronica atrophicans (ACA). Forty healthy blood donors served as controls. Four distinct antiphospholipid antibody ELISAs were developed, each using a different phospholipid coating: CL, PA, PC, and PS. Serum of APS patient was used as a positive control and for standard curve generation. Results: All four ELISAs were successfully established and demonstrated good measurement precision. Significant differences in antiphospholipid antibody levels and positivity rates were observed between Lyme borreliosis patients and healthy blood donors. Notably, levels of antibodies directed against PA (aPA), PC (aPC), and PS (aPS), both IgG and IgM, were significantly higher in patients with late Lyme borreliosis, manifested as ACA, compared to healthy blood donors. In contrast, anti-CL (aCL) levels did not differ significantly between groups. Patients with ACA also showed the highest frequency of multiple antiphospholipid antibody positivity, with 7 out of 10 patients testing positive for three or more antiphospholipid antibodies. Conclusions: Accurate and precise in-house ELISAs for the detection of aCL, aPA, aPC, and aPS using APS sera as standard material were developed and validated for the analysis of samples of patients with Lyme borreliosis. Our data suggest that antiphospholipid antibody levels—specifically aPA, aPC, and aPS—differ across clinical manifestations of Lyme borreliosis, with the greatest increases observed in patients with ACA.

Antibodies doi: 10.3390/antib15030050

Authors: Xin Rong Lim Ryan Xuan Wei Teo Rae Yi Xin Par Bernard Pui Lam Leung

Type I interferons (IFN-I), including IFN-α, IFN-β, and IFN-ω, are central to antiviral defence and immune regulation. Autoantibodies targeting IFN-I (anti-IFN-I AAbs) have emerged as key pathogenic factors in severe coronavirus disease 2019 (COVID-19) and are detectable in systemic lupus erythematosus (SLE), a prototypic IFN-driven autoimmune disease. Here we compare the prevalence and clinical impact of anti-IFN-I autoantibodies (Aabs) in COVID-19 and SLE based on a structured review of 53 studies from 2014 to 2025 and highlight the clinical associations and therapeutic opportunities presented by these autoantibodies. In COVID-19, neutralising anti-IFN-α and/or anti-IFN-ω AAbs were consistently associated with severe disease and impaired antiviral responses, particularly in older male populations. In SLE, anti-IFN-α AAbs were variably detected; neutralising antibodies were associated with reduced interferon gene signatures in some cohorts but inconsistent correlations with disease activity. Therapeutically, anti-IFN-I AAbs in COVID-19 may inform risk stratification and early antiviral strategies, whereas in SLE, IFN-α blockade, including IFN-α kinoid vaccination, demonstrates modulation of IFN signatures but variable clinical benefit. Notably, these findings reveal an immunological paradox: the same neutralising mechanism that impairs antiviral defence in COVID-19 may attenuate chronic IFN-driven inflammation in SLE. Taken together, anti-IFN-I AAbs exert context-dependent effects: pathogenic in acute viral infection yet potentially modulatory in chronic IFN-driven autoimmunity. Prospective longitudinal studies are required to further clarify their translational utility and long-term clinical impact.

Antibodies doi: 10.3390/antib15030049

Authors: Christian Caglevic Mario Alex Contreras-Torrez Felipe Reyes-Cosmelli Rodrigo Uribe-Maturana Mauricio Mahave Nicole Caire Luis Villanueva-Olivares Fernando Cid Alvaro Lladser Jorge Sapunar

Gallbladder cancer (GBC) is an aggressive tumor that, together with the cholangiocarcinomas, constitutes the spectrum of biliary tract cancer (BTC). These tumors are characterized by a frequently late diagnosis, marked genomic heterogeneity, variable response to cytotoxic therapies, and poor overall survival in advanced stages. Nevertheless, the characterization of the tumor microenvironment (TME) and the identification of actionable molecular targets have driven the development of biological therapies. This review summarizes current and emerging evidence on monoclonal antibodies, bispecific antibodies, and antibody–drug conjugates (ADCs) in the management of GBC. The analysis addresses the early exploration of autoantibodies as potential diagnostic biomarkers, mechanistic hypotheses of immune evasion, and the clinical translation of targeted agents in the metastatic setting. Additionally, we critically discuss the extrapolation of data from global BTC trials to the specific GBC setting, the integration of population genetics into epidemiological studies such as the EULAT Eradicate GBC initiative, and the preliminary status of immunotherapy in perioperative scenarios.

Antibodies doi: 10.3390/antib15030048

Authors: Francesco Ferrara Flavia De Berardinis Manlio Scognamiglio Andrea Zovi

Background: Monoclonal antibodies (mAbs) initially played a major role in outpatient COVID-19 management by providing rapid passive immunity and reducing progression to severe disease. However, continuous SARS-CoV-2 evolution progressively compromised the effectiveness of several anti-spike products. This narrative review summarizes the trajectory of COVID-19 mAbs across three phases: early clinical efficacy, loss of efficacy due to immune escape, and future directions. Methods: We conducted a narrative review focusing on mechanisms of action, pivotal clinical trials, and real-world effectiveness of neutralizing anti-spike mAbs and host-directed immunomodulatory mAbs. Emphasis was placed on the impact of variants—especially Omicron—on susceptibility and clinical use, as well as on emerging next-generation platforms. Results: First-generation neutralizing mAbs substantially reduced the hospitalization rates during the Alpha and Delta waves, while immunomodulatory mAbs became standard options for the hyperinflammatory phase in hospitalized patients. With the emergence of Omicron and its sub-lineages, extensive immune escape led to marked reductions in neutralization for many earlier anti-spike agents and consequent restrictions in use. Later-generation approaches targeting more conserved epitopes provided temporary solutions but were also challenged by ongoing antigenic drift. Host-directed immunomodulators retained clinical relevance because their mechanism is independent of viral spike mutations. Conclusions: The clinical role of monoclonal antibodies in COVID-19 has been dynamic and increasingly constrained by viral evolution. Future strategies should prioritize broadly neutralizing antibodies targeting conserved epitopes, innovative delivery platforms, and integration with real-time surveillance to preserve clinical utility in the endemic phase and improve preparedness for future outbreaks.

Antibodies doi: 10.3390/antib15030047

Authors: Victoria Sherwood Denise Harold Richard O’Kennedy Christine Loscher Paul Leonard

Monoclonal antibody (mAb) discovery has been transformed by advances in single-cell technologies, microfluidics, high-throughput sequencing, and computational design. Modern platforms enable the interrogation of large numbers of individual B cells, directly linking antibody sequence with antigen specificity and functional activity. Microfluidic and optofluidic systems now support high-throughput compartmentalisation and functional screening of antibody-secreting cells, while sequencing-based approaches allow parallel recovery of paired heavy- and light-chain sequences. These developments have shifted antibody discovery from binding-based selection toward function-first paradigms, enabling the rapid identification of diagnostic and therapeutically relevant antibodies. Integration with computational tools, including machine learning and structure-based modelling, has further enabled the emergence of closed-loop discovery pipelines, in which experimental and in silico methods iteratively refine candidates. This review summarises key advances in single-cell microtools over the last decade and highlights how the convergence of experimental and computational technologies is reshaping antibody discovery toward scalable, data-driven, and increasingly automated platforms.

Antibodies doi: 10.3390/antib15030046

Authors: Olga Strizhakova Grigory Poroshin Andrei Pershin Yana Bakhareva Ivan Shevchenko Ivan Lyagoskin Rakhim Shukurov Ravil Khamitov

Background/Objectives: Ocrelizumab is a humanized monoclonal antibody targeting CD20, approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). The neutralizing activity of anti-drug antibodies (ADAs), especially neutralizing ADAs (nADAs) activity, should be examined considering that it can alter pharmacokinetic (PK) and pharmacodynamic (PD) profiles, reduce drug efficacy, and lead to life-threatening adverse events. Methods: This article presents data on the development and validation of an assay for neutralizing anti-drug antibodies (nADA) based on ADCC reporter cells for the analysis of patient sera in the context of ocrelizumab clinical studies. Results: Critical steps and conditions to minimize assay variability were identified. The lower limit of detection was 549.6 ng/mL. The cutoff for nonspecific neutralization was determined as 19.7%. The presence of 0.37–3.0 μg/mL ocrelizumab in a biological sample enables the detection of 1.1–10.0 μg/mL polyclonal anti-ocrelizumab idiotype antibodies, respectively. Conclusions: The developed method can be used for immunogenicity studies of medicinal products containing ocrelizumab.

Antibodies doi: 10.3390/antib15030045

Authors: Nan Jia Weixiao Wang Guangwei Zhao Danfei Meng Liyuan Zheng Jinhua Dong

Background: The monkeypox virus (MPXV) has attracted considerable global attention due to its potential to cause widespread outbreaks, necessitating the development of rapid and accurate diagnostic methods of significant clinical importance. A29, a key envelope protein of MPXV, represents a promising diagnostic target. Methods: A novel monoclonal antibody, D10, was isolated from the human Tomlinson I+J phage display library by biopanning against the recombinant A29 protein. The D10 Fab fragment was expressed and purified, and its binding affinity was characterized by biolayer interferometry. Molecular docking was performed to predict potential interacting residues. Specificity and detection performance were evaluated by direct and competitive enzyme-linked immunosorbent assay (ELISA). Results: D10 possesses a unique complementarity-determining region sequence and exhibits strong binding affinity toward the A29 protein. Structural modeling analysis suggested potential interacting residues of A29, including Gln67, Arg74, Asn75, Arg81, and Asn84, which may primarily interact with Ser10, Thr5, Gly49, Gly47, and Glu97 in the heavy chain of D10. The binding affinity, determined by biolayer interferometry, showed a dissociation equilibrium constant of 6.44 nM, indicating strong binding capability. Furthermore, competitive ELISA demonstrated that D10 binds selectively to the A29 protein, with a half-maximal inhibitory concentration of 1.88 μg/mL and a limit of detection of 0.12 μg/mL. Conclusions: Overall, this monoclonal antibody provides a valuable tool for the immunological detection of MPXV and holds potential for future clinical diagnostic applications.

Antibodies doi: 10.3390/antib15030044

Authors: Chrysoula-Evangelia Karachaliou Evangelia Livaniou

Background/Objectives: Chronic spontaneous urticaria (CSU) is characterized by almost daily wheals or angioedema lasting for more than six weeks and not attributable to a defined inducing factor. CSU reportedly affects 1–2% of the general population and may lead to a substantial impairment in patients’ quality of life. Thus, developing methods that enable early diagnosis and assessment of disease activity is a major objective for scientists and clinicians. Methods: A significant proportion of CSU cases appears to be associated with autoimmune mechanisms, which mainly involve IgE autoantibodies (type I CSU), IgG autoantibodies (type IIb CSU), or both (type I and type IIb overlap). To this end, detection of specific IgE and/or IgG autoantibodies in CSU patients using biological or immunochemical assays can offer valuable information and enable further investigation and better management of the disease. Results: This review focuses on and presents various immunochemical assays, mainly ELISAs, for determining specific IgE and/or IgG autoantibodies, along with immunochemical methods for quantifying total IgE levels as an additional biomarker in CSU patients; the development and/or application of these assays has been reported in several papers published in the last decade on CSU. Conclusions: The methods presented have recently been applied and have substantially contributed to CSU diagnosis, endotyping and prediction of response to various treatments. Further validation of the existing immunochemical assays along with the development of reliable assays for novel autoantibodies and/or autoantigens will deepen our understanding of CSU pathogenesis and support the clinical diagnosis and treatment of CSU.

Antibodies doi: 10.3390/antib15030043

Authors: Carolyn Chiu Muhammad Zaki Jawaid Daniel Lee Cox

Background/Objectives: The unprecedented structural and binding data for antibodies to the SARS-CoV-2 virus taken together with the mutations for the spike protein allows for a broad simulation study of antibody–spike protein binding. This provides an understanding of the co-evolution of human immunity and viral immunity escape. Methods: We utilized the YASARA molecular dynamics program to generate initial structures and simulate to equilibration for six SARS-CoV-2 variants and ten different antibodies sampling two different binding regions to the receptor binding domain of the spike (especially for the Class I antibodies in the same part of the spike that attaches to the ACE2 receptor protein) and one to the N-terminal domain of the spike. Starting structures for antibody binding to variant spike protein domains are perturbatively achieved through point mutations and insertions/deletions in the YASARA program. We employed YASARA to measure interfacial hydrogen bound counts between antibodies and variant spike proteins and the HawkDock MMGBSA program to characterize trends in binding energies with mutation for four of the antibodies. We utilized the VMD program to analyze the time course of hydrogen bond populations. Results: As seen in previous studies, interfacial hydrogen bond counts serve as an excellent proxy for binding energies without the large systematic error inherent in the latter. We find that there is generally a decline in antibody binding strength, as measured by interfacial hydrogen bond counts, with viral evolution, but that a modest re-entrance of binding strength is present for most antibodies studied. Generically, the antibody heavy chain binds more strongly to the spike protein, though for approximately half the antibodies the light chain binding strength converges to the heavy chain strength with viral evolution. Conclusions: The key conclusion is that the identified re-entrant immunity, speculatively arising from a balancing of maintenance of ACE2-spike binding while escaping antibodies through mutation, allows for some maintenance and even strengthening of immunity for later viral strains from early infection or vaccination.

Antibodies doi: 10.3390/antib15030042

Authors: Radosław Łupkowski Karolina Górniak Maja Lisik-Habib Ewa Burchardt Radosław Mądry Monika Szarszewska Katarzyna Gabalewicz Dominika Pyszak Petra Bretova Beata Maćkowiak-Matejczyk Wioletta Sawczuk Monika Łączyńska-Madera Dagmara Klasa-Mazurkiewicz Angelika Gawlik-Urban Magdalena Michalik Zuzanna Borysiewicz Ewa Iwańska Mirosława Puskulluoglu Paweł Blecharz Renata Pacholczak-Madej

Background: Immunotherapy has become an integral part of systemic treatment for cervical cancer (CC). This study assessed the safety profile of cemiplimab and the association between immune-related adverse events (irAEs) and treatment outcomes in patients with persistent, recurrent or metastatic CC. Methods: This ambispective, multicenter, real-world cohort study included 101 patients treated in 13 reference oncology centers as part of the PCCIG-01 study. We evaluated the frequency and severity of irAEs and their association with progression-free survival (PFS) and overall survival (OS). Survival outcomes were analyzed using the Kaplan–Meier method and Cox proportional hazards models, with p < 0.05 considered statistically significant. Results: After a median follow-up of 7.5 months, adverse events occurred in 45 patients (44.6%) and were mostly grade (G) 1–2. IrAEs were observed in 34 patients (33.7%). Endocrine toxicities predominated (n = 24, 58.5% of irAEs), followed by hepatic (n = 5, 12.2%) and gastrointestinal events (n = 4, 9.8%). G3 irAEs occurred in 8 patients (7.9%). Median PFS was 3.9 months (95% CI 2.9–5.6) in patients without irAEs and 10.9 months (95% CI 5.7–16.3) in those with irAEs (p = 0.03). Median OS was 15.3 months (95% CI 8.6–25.9) in patients without irAEs and was not reached in those with irAEs (95% CI 11.6-NR; p = 0.11). The development of irAEs was associated with a 54% reduction in the risk of progression (HR 0.46, 95% CI 0.27–0.80), with no statistically significant impact on OS. Conclusions: In exploratory analyses, the occurrence of irAEs was associated with improved PFS in cemiplimab-treated patients with persistent, recurrent or metastatic CC. Cemiplimab showed a manageable safety profile, with most toxicities being G1–G2.

Antibodies doi: 10.3390/antib15030041

Authors: Alexandra Rak Veronika Muzurova Svetlana Donina Polina Prokopenko Irina Isakova-Sivak Larisa Rudenko

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems.

Antibodies doi: 10.3390/antib15030040

Authors: Giulio Turco Donatella Tarantino Antonietta Giuseppa Ferraro Giuseppina Greco Domenico Tricarico

Background/Objective: Follicular lymphoma (FL) is one of the most common indolent B-cell non-Hodgkin lymphomas (NHL) and is characterized by recurrent relapses despite advances in therapy. Bispecific antibodies that redirect T lymphocytes toward malignant B cells represent a major innovation in the treatment of relapsed or refractory disease. Mosunetuzumab is a CD20×CD3 bispecific antibody that induces T-cell mediated cytotoxicity against B-cell malignancies. In this manuscript, we describe the clinical experience with mosunetuzumab in three patients with relapsed or refractory FL treated at the Hospital Card. G. Panico, Tricase (LE). Methods: Clinical history, prior therapies, treatment responses, and safety outcomes are reported. Results: The cases illustrate the potential efficacy and manageable safety profile of mosunetuzumab in heavily pretreated FL patients. Conclusion: The effectiveness of this drug is confirmed in our center.

Antibodies doi: 10.3390/antib15030039

Authors: Kai Shimizu Hiroyuki Suzuki Mika K. Kaneko Yukinari Kato

Background/Objectives: Cadherin-13 (CDH13), part of the cadherin family, is attached to the plasma membrane through glycosylphosphatidylinositol. CDH13 plays essential roles in the development of the neurological and vascular systems and is a risk factor for neural and cardiovascular diseases. CDH13 is expressed on the plasma membrane in both mature and uncleaved precursor forms with the prodomain. Although several anti-CDH13 monoclonal antibodies (mAbs) are available for basic research, there have been no reports of anti-CDH13 mAbs that can detect both the mature form and the uncleaved precursor in flow cytometry. Methods: We developed novel anti-human CDH13 mAbs (named Ca13Mabs) using the mature form of CDH13-expressed cells as an antigen. Results: Among Ca13Mabs, a clone, Ca13Mab-17 (IgG2b, κ) specifically recognized the mature and uncleaved precursor CDH13-overexpressed Chinese hamster ovary-K1 (CHO/CDH13) cells with no detectable cross-reactivity toward 21 other cadherins by flow cytometry. Ca13Mab-17 also detected endogenous CDH13 in human glioblastoma (LN229 and U87MG) and lung mesothelioma (NCI-H2052) cell lines. The dissociation constant (KD) value of Ca13Mab-17 for LN229 was estimated at 4.1 × 10−8 M. Furthermore, Ca13Mab-17 detected both the mature and uncleaved precursor CDH13 in Western blotting. It also identified new blood vessels and glioblastoma cells by immunohistochemistry. Conclusions: Ca13Mab-17 is a versatile tool for detecting both mature and uncleaved precursor forms of CDH13 and has potential for tumor diagnosis and therapy.

Antibodies doi: 10.3390/antib15020038

Authors: Shanon T. Nizard Yiyang Chen Madhavika N. Serasinghe Ruben Fernandez-Rodriguez Kamrin D. Shultz Jesminara Khatun Anthony Mendoza Jesse D. Gelles Juan F. Henao-Martinez Ioana Abraham-Enachescu Md Abdullah Al Noman Stella G. Bayiokos J. Andrew Duty Shane Meehan Mihaela Skobe Jerry Edward Chipuk

Background/Objectives: Mitochondria are dynamic organelles that continuously undergo balanced cycles of fusion and division to maintain optimal function. Mitochondrial division is mediated by Dynamin-Related Protein 1 (DRP1), a cytosolic large GTPase whose phosphorylation at serine 616 (DRP1-S616Ⓟ) promotes its translocation to the outer mitochondrial membrane and organelle division. Dysregulated mitochondrial division disrupts cellular homeostasis and contributes to disease pathogenesis, including cancer. Our prior work demonstrated that the oncogene-induced mitogen-activated protein kinase (MAPK) pathway constitutively phosphorylates DRP1 at serine 616, which is essential to cellular transformation and correlates with oncogene status in patient tissues. Similarly, DRP1-S616Ⓟ is subject to pharmacologic control by targeted therapies against oncogenic MAPK signaling. Methods: Building upon this foundation, we developed and characterized a recombinant murine monoclonal antibody (referred to as 3G11) with high specificity for human DRP1-S616Ⓟ, raised against a peptide derived from the human DRP1 sequence. Results: Using diverse experimental platforms, we demonstrate the robust utility of 3G11 to detect DRP1-S616Ⓟ in melanoma cell extracts and isolated organelles. Immunofluorescence revealed that pharmacologic inhibition of oncogenic MAPK signaling reduces DRP1-S616Ⓟ levels, which correlates with mitochondrial hyperfusion, while immunohistochemistry showed that elevated DRP1-S616Ⓟ expression in human tissues correlates with BRAFV600E disease. Conclusions: 3G11 is a new recombinant antibody for detecting DRP1-S616Ⓟ and supports studies of mitochondrial division in cancer. Together, these findings establish 3G11 as a specific, versatile, renewable, and cost-effective tool for studying mitochondrial division, with strong potential for clinical applications.

Antibodies doi: 10.3390/antib15020037

Authors: Rachita K. Sumbria Ruben J. Boado

Antibodies directed against β-amyloid (Aβ) have been developed for the treatment of Alzheimer’s disease (AD). However, the in vivo central efficacy is reduced by the poor penetration of antibodies across the blood–brain barrier (BBB). In addition, these antibodies have been associated with adverse effects like amyloid-related imaging abnormalities. Thus, the development of new antibody-based therapies for AD with improved transport across the BBB may improve efficacy and reduce adverse effects. Antibodies targeting the BBB transferrin receptor (TfR) are able to cross the BBB through receptor-mediated transcytosis, producing a global distribution throughout the brain. Along the same line, bispecific antibodies directed to both the BBB TfR and Aβ showed enhanced brain uptake and pharmacological effects with diminished adverse side effects in experimental animal models of AD and in clinical trials. A generation of brain-penetrating fusion proteins targeting the BBB-TfR has been shown to represent novel treatments for AD, and this includes erythropoietin, tumor necrosis factor alpha inhibitors, neprilysin, somatostatin, oligonucleotides, and an antibody activating TREM2. The aim of this article is to review the progress made in the delivery of antibody-derived biologicals to the brain for AD, targeting the BBB-TfR.

Antibodies doi: 10.3390/antib15020036

Authors: Katarzyna Pogoda Karolina Lewińska Paulina Kalman Anna Bałata Piotr J. Wysocki

The treatment paradigm for HER2-positive metastatic breast cancer has evolved from continuous chemotherapy-based regimens to a model of finite chemotherapy induction followed by sustained antibody-driven disease control. The CLEOPATRA trial established dual HER2 blockade with trastuzumab and pertuzumab plus a taxane as the biological and clinical anchor of this approach, demonstrating that chemotherapy is administered for a defined induction period, after which antibody maintains disease suppression. An increasing body of clinical evidence indicates that antibody-based regimens can be combined with targeted agents, including CDK4/6 inhibitors or HER2 tyrosine kinase inhibitors, to achieve durable disease control without the need for continuous chemotherapy. In the PATINA trial, the addition of palbociclib to trastuzumab, pertuzumab, and endocrine therapy was associated with a clinically meaningful improvement in progression-free survival in hormone receptor-positive, HER2-positive metastatic breast cancer. At the same time, quality of life was maintained despite higher rates of hematologic toxicity. More recently, HER2-CLIMB-05 demonstrated that the addition of tucatinib to dual HER2 antibody therapy significantly prolonged progression-free survival, supporting a model of sustained, multi-agent HER2 pathway suppression. The monarcHER trial provided biological proof of concept that antibody plus CDK4/6 inhibition can achieve disease control without chemotherapy in hormone receptor-positive, HER2-positive disease. Collectively, these advances support a translational framework in which antibody therapy serves as a central component of treatment strategies, with targeted partners selected according to tumor biology and prior therapy. This review summarizes the biological basis, clinical evidence, and future perspectives of antibody-driven maintenance in HER2-positive metastatic breast cancer.

Antibodies doi: 10.3390/antib15020035

Authors: Marie-Paule Lefranc Gérard Lefranc

The immunoglobulins (IG) or antibodies and the T cell receptors (TR) are the antigen receptors of the adaptive immune responses (AIR) of jawed vertebrates (Gnathostomata). IMGT®, the international ImMunoGeneTics information system®, was created in 1989 by Marie-Paule Lefranc (Laboratoire d’ImmunoGénétique Moléculaire (LIGM), Université de Montpellier and CNRS) to deal with and to manage the huge diversity of IG or antibodies and TR. The founding of IMGT® marked the advent of immunoinformatics, a new science which emerged at the interface between immunogenetics and bioinformatics. For the first time, the IG and TR variable (V), diversity (D), joining (J) and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. The IMGT-ONTOLOGY CLASSIFICATION axiom and the concepts of classification have generated the IMGT nomenclature and the IMGT Scientific chart rules for assigning IMGT names to IG and TR genes and alleles of Homo sapiens and of any other jawed vertebrate species. The IMGT nomenclature is used for genes in locus, in sequences (genomic or rearranged, expressed or not) and in structures enabling comparative immunology, evolutionary immunogenetics, standardized analysis and comparison of IG and TR repertoires analysis in normal or pathologic situations. IMGT nomenclature is used in basic, veterinary, and medical research, in clinical applications (mutation analysis in leukemia and lymphoma), and in therapeutic antibody design, engineering and humanization. By providing consistent and high standard biocuration for the description of the IG and TR loci, genes and alleles, and for the analysis of the IG or antibody and TR-expressed rearranged sequences and proteins and structures, the IMGT nomenclature is the common language for immunoinformatics and artificial intelligence (AI).

Antibodies doi: 10.3390/antib15020034

Authors: Tingting Ji Zhaoyun Zong Ningyuan Gong Minghui Yan Shiyu Chen

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (KD = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, KD = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems.

Antibodies doi: 10.3390/antib15020033

Authors: Maksymilian Markwitz Natalia Welc Klementyna Kępińska Monika Bowszyc-Dmochowska Marian Dmochowski

Background: Pemphigus diseases are rare autoimmune blistering disorders mediated by pathogenic autoantibodies directed mainly against desmoglein 1 and desmoglein 3. Although most cases are considered idiopathic, external triggers that can disrupt immune tolerance have been described. Vaccination has been discussed as a potential precipitating factor in autoimmune skin diseases. However, the relationship between vaccination and the induction of pemphigus-related autoantibodies has not been comprehensively summarized. Methods: We conducted a narrative review of all available studies published in the last 25 years identified through medical databases, excluding studies on COVID-19 vaccinations. Reports describing either new-onset pemphigus or exacerbation of preexisting pemphigus with a temporal association to vaccination were included. Clinical characteristics, vaccine type, latency period, direct immunofluorescence findings, and ELISA results for desmoglein autoantibodies were analyzed. In addition, we present our own clinical-laboratory experience illustrating this issue. Results: The current evidence consists predominantly of case reports and small case series. Published cases describe pemphigus vulgaris and pemphigus foliaceus occurring after vaccinations against influenza, hepatitis B, tetanus, diphtheria, pertussis, rabies, and other routinely administered immunizations. The latency period most often ranged from several days to a few weeks. Immunopathological findings were consistent with classical pemphigus diseases, including intercellular IgG deposits in the epidermis and circulating autoantibodies against desmoglein 1 and/or desmoglein 3. Our patient was a 78-year-old woman who developed cutaneous form of pemphigus vulgaris, diagnosed with direct immunofluorescence (DIF) and multiplex ELISA, 10 days after diphtheria–tetanus–pertussis vaccination. The patient had a positive family history of autoimmune blistering disease, namely mucous membrane pemphigoid. Conclusions: Based on the currently available evidence, a direct causal relationship between vaccination and pemphigus diseases cannot be established. Nevertheless, accumulated clinical and serological observations suggest that vaccination may act as a triggering factor in genetically or immunologically predisposed individuals, possibly by amplifying pre-existing subclinical autoreactive immune responses. Further population-based and mechanistic studies are required to clarify this association, while the overall benefits of vaccination remain substantial.

Antibodies doi: 10.3390/antib15020032

Authors: Chiara Orlandi Angela Tincani Micaela Fredi Laura Andreoli Francesca Crisafulli Liala Moschetti Cecilia Nalli Maria Grazia Lazzaroni Marco Taglietti Matteo Filippini Sonia Zatti Laura Picciau Franco Franceschini Ilaria Cavazzana

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women of childbearing age, and active disease during pregnancy is associated with increased maternal and fetal morbidity. Belimumab is an effective biologic therapy for active SLE; however, its use during pregnancy has long been limited by the scarcity of safety data. Recent evidence and updated international recommendations suggest that belimumab may be considered in selected cases when required to maintain maternal disease control. We report the case of a woman with SLE who experienced three consecutive pregnancies with live births between 2019 and 2024 while receiving belimumab, allowing an intra-individual comparison of different exposure strategies. During the first pregnancy, belimumab was discontinued at conception and was followed by a disease flare in late pregnancy and postpartum. In the second and third pregnancies, belimumab was continued until gestational week 20 following shared decision-making with the patient; nevertheless, disease flares occurred during the third trimester of both pregnancies. All pregnancies resulted in live births at term, with no congenital anomalies, placental insufficiency, or fetal growth restriction. One neonate from the third pregnancy developed early-onset neonatal sepsis and meningitis, which resolved completely after antibiotic treatment. All children are currently growing and developing normally. This case supports a risk-adapted approach to belimumab use during pregnancy. In selected women with SLE at high risk of disease reactivation, continuation of belimumab until mid-gestation may contribute to improved maternal disease control without evident adverse fetal outcomes.

Antibodies doi: 10.3390/antib15020031

Authors: Julia Cieślawska Mariola Pawlaczyk Justyna Gornowicz-Porowska

Human hair performs a number of important physiological and esthetic functions. Hair loss and alopecia are complex disorders which affect people all over the world. Hair loss can be an early manifestation of various autoimmunological disorders. Despite a growing interest of researchers in the role of immune factors—especially autoantibodies—in the etiology of certain types of alopecia, their role in alopecia remains uncertain. Several potential autoantigens of follicular components, mainly derived from keratinocytes and melanocytes of the hair follicles, have been found to play a role in the development of alopecia areata. The list of autoantigens includes trichohyalin, keratin 16, fibroblast growth factor receptor 3, glycoprotein-100, melanoma-associated antigen recognized by T cells 1, dopachrome tautomerase/tyrosinase-related protein 2, tyrosinase, and tyrosine hydroxylase. This narrative review presents different aspects of immunopathogenesis of alopecia, from physiology (hair follicle immune privilege) to pathology (disruption of hair follicle immune privilege) and signaling pathways. Identification of key autoantigens could potentially pave the way for the development of new, effective, and more targeted immunotherapies for alopecia.

Antibodies doi: 10.3390/antib15020030

Authors: Sharon L. Walmsley Leif Erik Lovblom Bryan Boyachuk Curtis Cooper Valérie Martel-Laferrière Mona Loutfy Marie-Louise Vachon Shariq Haider Pamela Aldebes Karen Colwill Anne Claude Gingras Freda Qi Marina B. Klein

Objectives: To determine the incidence and outcomes of SARS-CoV-2 infection and to evaluate seroconversion rates and quantify antibody responses to COVID-19 vaccines in two cohorts of persons living with HIV at a possible higher risk of poor outcomes (HCV coinfection and those over the age of 65 years). Methods: We included participants from two established cohorts of persons living with HIV, those who were older than 65 years of age, and those with hepatitis C (HCV) co-infection. Four hundred and seventy-one participants completed questionnaires on SARS-CoV-2 infection and COVID-19 vaccine doses and submitted peripheral blood specimens for measuring antibody levels to COVID-19 antigens, full-length spike trimer, its receptor binding domain (RBD), and nucleocapsid protein (N) at 6-month intervals up to three visits between February 2021 and December 2024. Logistic and ordinal logistic regression models evaluated predictors of seroconversion and antibody levels. Results: Overall, 51% of participants developed a SARS-CoV-2 infection, but it was mild, with only nine requiring hospital admission and no deaths. Overall, 99% of tested specimens had antibodies above threshold to either spike or RBD proteins. Specimens that did not and those with lower antibody levels had testing earlier in the pandemic, and were from participants with fewer vaccine doses, and did not have natural infection. Age, depression, comorbidity, HCV co-infection, current substance use, CD4 count, or HIV viral load were predictive of antibody level. Those with hybrid immunity had higher antibody responses. Conclusions: In cohorts of persons with HIV-HCV coinfection and those who are ageing, we observed high rates of seroconversion to COVID-19 antigens. Antibody levels were higher among those with more vaccine doses, hybrid immunity, and later in the pandemic waves. Although 51% developed a breakthrough infection, outcomes were mild with no deaths.

Antibodies doi: 10.3390/antib15020029

Authors: Beata Pamuła Martyna Banach Marta Mikońska Karolina Korytkowska Krzysztof Lacek Oliwia Śniadała Małgorzata Marczak Krzysztof Flis Aleksandra Sowińska Damian Kołakowski Jerzy Pieczykolan Beata Zygmunt Maciej Wieczorek Olga Abramczyk

Background/Objectives: Antibodies are a rapidly expanding field in drug discovery, but their monospecificity limits therapeutic applications, particularly in complex inflammatory diseases. Multispecific therapeutics, which combine variable regions targeting two or more antigens, offer potential advantages such as enhanced efficacy, broader target modulation, and reduced side effects. This study aimed to identify and characterize bispecific, VHH-based antibodies simultaneously targeting IL-6 and IL-17A—two key cytokines involved in autoimmune and chronic inflammatory conditions. Methods: A phage display screening was conducted using llama-derived VHH libraries to select binders against human IL-6 and IL-17A. Binding affinities of individual VHHs and assembled bispecific constructs were assessed using Bio-Layer Interferometry (BLI). Functional activity was evaluated using reporter cell lines responsive to IL-6 and IL-17A signaling. Biophysical and quality assessments of selected VHHs and bispecific antibodies were performed using the Uncle screening platform and LabChip capillary electrophoresis. Results: Several high-affinity VHH binders were identified for both IL-6 and IL-17A, and incorporated into bispecific antibody formats. The bispecific candidates exhibited simultaneous inhibition of both cytokine pathways in functional reporter assays. Biophysical characterization confirmed good stability and purity profiles for selected molecules. Conclusions: This study demonstrates the feasibility of generating stable, functional bispecific VHH-based antibodies targeting IL-6 and IL-17A. These constructs show potential as therapeutic agents for treating autoimmune and chronic inflammatory diseases by modulating multiple signaling pathways simultaneously.

Antibodies doi: 10.3390/antib15020028

Authors: Maddalena Calvo Marta Caccamo Dalila Maria Cammarata Laura Trovato

Invasive candidiasis (IC) remains a significant cause of morbidity and mortality among critically ill, hematologic, and neonatal patients worldwide. Rapid and accurate diagnosis is essential to guide timely antifungal therapy and improve outcomes. Among available diagnostic tools, 1,3-β-D-glucan (BDG), a polysaccharide component of the fungal cell wall, has emerged as a key biomarker. BDG assays allow for early detection of probable IC, often preceding positive blood cultures, and offer prognostic information based on serial measurements. Species-specific differences in Candida cell wall composition influence BDG release and diagnostic sensitivity. Candida albicans generally correlates with high BDG levels, whereas Nakaseomyces glabrata, Candida parapsilosis, and Candida auris exhibit variable or lower glucan exposure, limiting assay sensitivity. BDG performance is affected by patient-specific factors, such as prior surgery, transfusions, or coexisting bacterial infections, which may lead to false-positive results. Molecular techniques, including PCR-based assays, provide complementary diagnostic accuracy and species identification, and their combination with BDG testing enhances sensitivity up to 90%. Serial BDG monitoring supports risk stratification and treatment response assessment, with persistent elevations predicting worse outcomes. In neonatal and pediatric populations, optimal cut-off values remain under investigation, highlighting the need for integration with clinical and microbiological data. Overall, BDG represents a valuable adjunct in a multimodal diagnostic workflow, providing both diagnostic and prognostic insights in invasive candidiasis management.

Antibodies doi: 10.3390/antib15020027

Authors: Sanika Naware Saurav Kulkarni Sahil Salvi Dhvani Patel Dhaval K. Shah

Background/Objectives: The neonatal Fc receptor (FcRn) plays a crucial role in extending the systemic half-life of monoclonal antibodies (mAbs), but its influence on ocular distribution remains incompletely understood. This study investigated the impact of FcRn on the ocular disposition of mAbs following systemic administration in rabbits. Methods: New Zealand White rabbits received a single intravenous dose (1 mg/kg) of either wild-type trastuzumab (TS-WT) or its FcRn non-binding variant (IHH). Plasma and ocular tissues (retina, iris–ciliary body, vitreous humor, aqueous humor, cornea, conjunctiva, and tears) were collected at terminal time points up to 336 h for TS-WT and 168 h for IHH. Antibody concentrations were quantified using a validated sandwich ELISA. Pharmacokinetic parameters and antibody biodistribution coefficients (ABC) were calculated to assess the FcRn-mediated effects on ocular distribution. Results: TS-WT demonstrated 2-fold higher systemic exposure compared to IHH. The iris–ciliary body exhibited the highest absolute exposure for both antibodies, with TS-WT showing significantly higher accumulation (ABC0–168h: 14.95% vs. 8.89%). Retinal distribution remained comparable between antibodies (5.96% vs. 5.51%). Both antibodies were detectable in tears, with ABC value of ~4% reported for TS-WT. TS-WT also demonstrated markedly increased distribution in vitreous humor and tear fluid (3.5- and 5.5-fold higher ABC values, respectively) compared to IHH. The cornea (5.76% vs. 5.57%) and conjunctiva (7.71% vs. 7.21%) showed comparable relative distribution between TS-WT and IHH, while aqueous humor showed minimal differences (0.44% vs. 0.52%). Conclusions: This investigation reveals distinct tissue-specific patterns of FcRn-mediated mAb distribution within the eye. FcRn binding significantly enhanced antibody distribution in ocular tissues, such as the iris–ciliary body, and tears, with less pronounced effects on the retina, cornea, conjunctiva and aqueous humor. These findings provide mechanistic insights for optimizing mAb-based therapeutics for ocular disease and understanding the ocular toxicity of mAb-based therapeutics, such as antibody–drug conjugates.

Antibodies doi: 10.3390/antib15020026

Authors: Jingming Zhang Matthew Larsen Timothy Blanc Babita S. Parekh Ming-Ching Hsieh

Background: Protein A resins are indispensable for monoclonal antibody (mAb) production, yet their condition and performance are traditionally assessed using indirect or qualitative methods. In this study, the multi-attribute method (MAM), previously applied to therapeutic protein characterization, is systematically adapted for the first time as a unified liquid chromatography–mass spectrometry (LC-MS) platform for Protein A resin analysis. Method: Four Cytiva Protein A resins, MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, and MabSelect™ PrismA, were evaluated by MAM for resin identity, Protein A ligand integrity, fouling by impurities, and cleaning performance. Results: MAM enables resin-specific peptide fingerprinting and quantitative monitoring of Protein A ligand post-translational modifications (PTMs), including deamidation, isomerization, and fragmentation induced by repeated clean-in-place (CIP) cycles. Comparative analysis of virgin and used resins revealed ligand degradation and fouling despite engineered alkaline stability, with MabSelect™ showing the greatest susceptibility. Importantly, residual monoclonal antibodies (mAbs) and host cell proteins (HCPs) were directly detected and quantified from the resin matrix, providing a molecular-level assessment of resin cleaning effectiveness not achievable with conventional approaches. Conclusions: This work establishes MAM as a novel, sensitive, and comprehensive strategy for Protein A resin lifecycle management, delivering actionable insight for resin selection, cleaning optimization, and downstream process development.

Antibodies doi: 10.3390/antib15020025

Authors: Davide Bagnara Andrea Nicola Mazzarello Monica Colombo Ennio Nano Niccolò Cardente Fabiana Ferrero Nadia Bertola Vanessa Cossu Fabio Ghiotto Adalberto Ibatici Emanuele Angelucci Antonino Neri Massimo Gentile Fortunato Morabito Manlio Ferrarini Giovanna Cutrona Franco Fais

Background/Objectives: The immunoglobulin heavy-chain variable (IGHV) gene repertoire represents a characteristic feature of chronic lymphocytic leukemia (CLL), although its configuration is not well defined at the early disease stages. The IGHV repertoire of a cohort of early CLL patients was analyzed and compared to that of a “real-world” reference cohort. Methods: Patients from the O-CLL1 observational protocol, which enrolled only Binet stage A cases within twelve months from diagnosis, were studied. IGHV/IGHJ rearrangements were sequenced and annotated following ERIC recommendations, and stereotyped subsets were assigned using ARResT/AssignSubsets. The repertoire features were compared with the dataset of a real-world cohort of patients with heterogeneous staging (CTR cohort) and with published early-diagnosis series. Results: IGHV and IGHJ gene distributions and HCDR3-length profiles in O-CLL1 closely mirrored those of CTR, indicating that the BcR IG repertoire at diagnosis is already defined rather than being selected during disease progression. Mutated IGHV (M-CLL) predominated, with a frequency of stereotyped BcR IG comparable to that of other early-diagnosis cohorts. However, within this conserved framework, subset #4 was over-represented among M-CLL from O-CLL without an increased overall IGHV4-34 gene usage, suggestive of a selective expansion rather than a recombinational bias. Subset #4 cases retained canonical HCDR3 motifs and showed time-to-first-treatment like other M-CLL, likely reflecting the younger age structure of O-CLL1. Conclusions: Early-diagnosis CLL displays a biased IGHV repertoire with stereotyped configurations characteristic of CLL, including subsets that are rare in the normal B-cell repertoire. These findings support a central role for antigen-driven selection in shaping CLL evolution.

Antibodies doi: 10.3390/antib15020024

Authors: Maurizio Benucci Elisa Cioffi Francesca Li Gobbi Emanuele Antonio Maria Cassarà Riccardo Terenzi Edda Russo Valentina Grossi Barbara Lari Maria Infantino Mariangela Manfredi

COVID-19 mRNA vaccines activate type I interferon pathways and in genetically or immunologically predisposed individuals may trigger autoimmune responses, including autoantibodies against melanoma differentiation-associated protein 5 (MDA5). Although cases of dermatomyositis (DM), particularly anti-MDA5-positive DM, have been increasingly reported after SARS-CoV-2 vaccination, its clinical spectrum and management remain incompletely defined. We conducted a narrative review of the literature on post-vaccination dermatomyositis, focusing on clinical features, autoantibody profiles, therapeutic approaches, and outcomes. The review was enriched by the inclusion of a new case: a 60-year-old woman who developed anti-MDA5-positive dermatomyositis two weeks after receiving her fourth dose of the BNT162b2 (Pfizer/BioNTech) vaccine. She presented predominantly with cutaneous and articular manifestations in the absence of interstitial lung disease. Treatment with oral prednisone, intravenous alprostadil, and the Janus kinase inhibitor tofacitinib resulted in marked clinical improvement. This case, together with the literature review, illustrates both typical and atypical presentations of vaccine-associated anti-MDA5 DM, highlights diagnostic challenges without lung involvement, and suggests JAK inhibition as a potential therapeutic option, contributing to a more comprehensive understanding of post-vaccination dermatomyositis.

Antibodies doi: 10.3390/antib15020023

Authors: Massimo Papale Carmela Paolillo Loredana Iafelice Tiziana Trivisano Giuseppe Stefano Netti Elena Ranieri Gaetano Corso

Background: Anti-double-stranded DNA (anti-dsDNA) antibodies are a key serological marker for systemic lupus erythematosus (SLE) and are commonly assessed in conjunction with anti-nuclear antibody (ANA) testing by indirect immunofluorescence (IIF) on HEp-2 cells. However, their detection is influenced both by the heterogeneity of the autoimmune response and by the characteristics of the analytical method employed, thereby complicating diagnostic interpretation. Methods: In this retrospective single-center study, 3090 consecutive patients undergoing anti-dsDNA analysis were screened, and 138 positive individuals, with anti-dsDNA levels ≥ 15 IU/mL by fluoroenzyme immunoassay (FEIA), were included in the study. A control group of 29 anti-dsDNA-negative patients was also analyzed. Anti-dsDNA-positive patients were stratified by antibody level (low, mild, high), and the results were correlated with HEp-2 IIF titers and fluorescence patterns. Furthermore, in a subset of 30 positive patients, anti-dsDNA antibodies were evaluated using immunoblotting (IB) and the Crithidia luciliae indirect immunofluorescence test (CLIFT). Statistical analyses assessed associations and concordance among methods. Results: Higher anti-dsDNA levels were generally associated with higher HEp-2 IIF titers. However, a considerable percentage (35%) of patients with positive anti-dsDNA were negative by HEp-2 IIF. Notably, high anti-dsDNA levels were detected in 19% of HEp-2 IIF-negative patients (titer < 1:80), 18% of mildly HEp-2 IIF-positive patients (titer 1:80–1:160), and 25% of HEp-2 IIF-positive patients (titer > 1:320). In the subset of 30 positive patients, FEIA analysis showed high concordance with the immunoblot in both IIF-positive (81%) and -negative (100%) patients, while CLIFT demonstrated lower agreement with both FEIA and IB independently of the IIF. Conclusions: Our findings indicate that anti-dsDNA antibody detection may occur independently of HEp-2 IIF positivity and that FEIA, especially when confirmed by immunoblot, represents a reliable approach for anti-dsDNA assessment. The observed results in this study likely reflect differences in epitope recognition and assay sensitivity among methods, suggesting the use of a multi-step diagnostic strategy in the serological evaluation of SLE.

Antibodies doi: 10.3390/antib15020022

Authors: Philipp D. Kaiser Simon Straß Sandra Maier Evgenia Herbold Bjoern Traenkle Anne Zeck

Background/Objectives: Developability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here, we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. Methods: Assays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency (“chelatability”). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze–thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimize experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. Results: A tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. Conclusions: This study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules.

Antibodies doi: 10.3390/antib15020021

Authors: Marcos García-Ocaña Lorea Legazpi-Olabide Sandra Rodríguez-Rodero Paula Rodríguez-Folgueira Iván Fernández-Vega Marcos Ladreda-Mochales Juan R. de los Toyos Luis J. García-Flórez

Background: Collagen XIα1, encoded by the COL11A1 gene, is a minor fibrillar collagen that is overexpressed in various human cancers, in which its presence correlates with tumor aggressiveness and progression. Methods: In this study, we developed two novel mouse monoclonal antibodies (mAbs)—anti-colXIα1 clone 3 and anti-colXIα1 clone 9—that target the putative C-telopeptide of human collagen XIα1. These antibodies target the RRHTEGMQA sequence, a unique nine-amino-acid stretch within the putative C-telopeptide of human collagen XIα1. Results: Corresponding to nearly identical V(D)J gene segments and complementarity-determining regions (CDRs), the antibodies specifically bound the RRHTEGMQA epitope in ELISAs but did not react with the C-propeptide. This specificity was further confirmed with the purified anti-colXIα1 clone 9 mAb, which demonstrated strong reactivity against recombinant proteins containing the RRHTEGMQA sequence in both ELISAs and Western blot assays. This sequence seems to behave as a linear B-cell neoepitope, in which the RRHT motif is crucial for epitope recognition. Otherwise, no immunodetections were observed, either in cultures and lysates from the COL11A1-highly expressing A204 human cell line or on tissue sections from specimens of human pancreatic ductal adenocarcinoma (PDAC), with strong desmoplastic reactions. Conclusions: Given the lack of precise knowledge of the characteristics of the putative C-telopeptide of human collagen XIα1, the presented antibodies could enhance our understanding of the processing of human procollagen XIα1 and contribute to better characterization of the tumor microenvironment of COL11A1-expressing cancers.

Antibodies doi: 10.3390/antib15020020

Authors: Maja Vilibić Klara Barbić Maja Bogdanić Snježana Židovec-Lepej Ana Matošić Ana Sanković Dalibor Karlović Leona Radmanić Matotek Nataša Kutela Sergej Nadalin Ema Borko Vladimir Savić Ljubo Barbić Marija Santini Hrvojka Janković Vladimir Stevanović Tatjana Vilibić-Čavlek

Background/Objectives: Viral hepatitis A–E represents a significant public health problem. Data on the prevalence of viral hepatitis markers among alcoholics are inconsistent. Methods: The study included 151 patients treated for alcohol abuse in one Croatian center. The control group consisted of 110 individuals from the general population tested for a routine check-up. The prevalence of viral hepatitis markers was assessed using serology and molecular methods. Results: The prevalence rates of hepatitis markers among patients were as follows: anti-HAV, 15.2%; anti-HBs, 11.9%; anti-HBc/anti-HBs, 2.6%; anti-HCV, 4.0%; and anti-HEV, 14.6%. HCV RNA was detected in one patient (0.6%). Compared with the control group, patients showed significantly higher HCV seroprevalence (4.0 vs. 0%), while the prevalence of other hepatitis markers did not differ significantly between the groups. The anti-HAV prevalence was associated with age (from 0% in patients aged <40 years to 42.9% in patients aged 60+ years), employment status (highest among retired individuals at 46.2%), and age of occasional alcohol consumption (highest seroprevalence of 26.3% in those who reported consumption between 22 and 25 years). The association between anti-HEV and educational level was of borderline significance. Logistic regression showed that older and retired patients and those who consumed alcohol occasionally between 22 and 25 years showed higher odds for HAV seropositivity (OR = 11.454–49.400, OR = 6.857, and OR = 4.464, respectively). Patients with university degrees were at lower risk for HEV seroprevalence (OR = 0.083). Conclusions: Alcoholic patients showed a higher HCV seroprevalence than the general population, while the prevalence of other viral hepatitis markers did not differ between the groups. Further studies on a larger cohort of patients are needed to confirm these findings.

Antibodies doi: 10.3390/antib15020019

Authors: Yunfeng Liu Qiting Huang Dongna Zhang Yingjun Wang Shuaiying Zhao Jianchuan Wen Yingying Kong Jianfeng Xu

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies.

Antibodies doi: 10.3390/antib15020018

Authors: Isamu Tsuji Kumiko Okada Benjamin Kroppen Tetsufumi Katta Kaori Yamamura Takeshi Nishihama Ayako Miura Hansjörg Götzke Eric Crampon Andrea Bertolotti-Ciarlet

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing.

Antibodies doi: 10.3390/antib15010017

Authors: Sébastien Estaran Bernard Hehlen Alain Chavanieu

Background/Objectives: Antibody-dependent cellular cytotoxicity relies on the interaction between the Fc region of immunoglobulin G1 (IgG1) and the CD16a receptor. While removal of core fucosylation on Fc and introduction of the DFTE mutation set (S239D, H268F, S324T, I332E) are known to enhance CD16a binding, the detailed contributions of these engineered sites in solution remain incompletely defined. Methods: Here, we employed 1 µs molecular dynamics simulations to map, at atomic resolution, the interaction networks stabilizing pre-formed Fc-CD16a complexes, including afucosylated Fc-wild-type, DFTE-engineered, Fc-fucosylated, and asymmetrically engineered Fc variants. Results: Our results show that only S239D, present on both Fc chains, and H268F on chain A consistently contribute to stabilizing the CD16a interface, while I332E does not form persistent interactions. Glycan–protein contacts are primarily intrachain, with transient interchain glycan–glycan interactions not contributing significantly to complex stability. Fucosylation on Fc significantly reduces binding stability by disrupting peripheral interactions and critical glycan-mediated contacts. Notably, the asymmetric Fc variant, in which the two heavy chains carry distinct sets of substitutions, retains high-affinity binding despite lacking S239D and carrying core fucose, through a novel hydrophobic cluster and reinforced peripheral electrostatic interactions. Conclusions: Altogether, these findings provide a quantitative framework for how targeted mutations and fucose modifications remodel Fc-CD16a interactions, offering insights for the rational design of next-generation therapeutic antibodies.

Antibodies doi: 10.3390/antib15010016

Authors: Juan Carlos Silva-Espinoza Mauricio I. Rodriguez Rodriguez Claire Eunise Perucho Brian A. Terrazas Carlos Valenzuela Stephany Palos Vargas Andrea Carlin Diana L. Prospero Giulio Francia Manuel Llano

Background/Objectives: Schlafen (SLFN) 8 and SLFN9 are mouse members of the Schlafen protein family, believed to have arisen through a gene duplication event. The physiological roles of these proteins remain poorly defined, in part due to the absence of reliable, commercially available antibodies for their detection. Methods: To develop specific antibodies, we performed an amino acid sequence alignment of these proteins and identified a thirteen amino acids long peptide predicted by AlphaFold modeling and hydropathicity analysis to be surface-exposed in both SLFN proteins. The SLFN8 peptide was conjugated to KLH and used to immunize mice, employing Poly(I:C) as an adjuvant. Results: We verified the anti-SLFN8 antibody specificity in mouse tissues, engineered human cells, and recombinant proteins by different immunodetection techniques, including Western blotting, immunoprecipitation, immunohistochemistry, and ELISA. Furthermore, splenocytes from immunized mice were used to generate hybridomas that secreted IgG antibodies with SLFN8-peptide specificity, as assumed by ELISA. Conclusions: Our results demonstrate that the identified peptide is highly immunogenic and capable of eliciting antibodies that distinguish between these two exceedingly similar proteins in a broad group of immunodetection techniques.

Antibodies doi: 10.3390/antib15010015

Authors: Aya Jalil Nadia Touil Omar Nyabi Elmostafa El Fahime Sara Benlhachemi Jean-Luc Gala Khalid Ennibi Karim Bakkouri Abdelaziz Benjouad Lamiae Belayachi

Background: bacterial pathogens and their toxins present analytical challenges for rapid and specific detection, contributing to over 600 million cases of illness annually and worsening antimicrobial resistance (AMR). Conventional detection methods are useful but limited. Single-domain antibodies (sdAbs) offer alternative recognition elements with unique biochemical and engineering benefits, enabling the development of nanobody-based immunoassays and biosensing platforms that provide fast, highly selective, and reliable detection of bacterial pathogens and toxins in both food and clinical environments. Objectives: this systematic review assesses the analytical and functional performance of nanobody-based immunoassays and sensing formats for detecting bacteria and toxins across food and clinical samples. Methods: following PRISMA guidelines, major scientific databases were used to gather research, resulting in 32 eligible studies published between 2011 and 2025. Results: data collected included assay platforms, target bacteria and toxins, limit of detection, sensitivity, specificity, matrix recovery, and practicality. Risk of bias was evaluated using an adapted QUADAS-2 framework. The review shows that nanobody-based immunoassays have achieved high performance, thermostability, compatibility with genetic engineering, and versatile assay design. When combined with advanced transduction and signal amplification strategies, these systems contribute to the development of highly sensitive and user-friendly bioanalytical platforms for detecting bacteria and toxins. Conclusions: however, most studies relied on spiked samples and lacked large-scale validation, emphasizing the need for standardized benchmarking and real-world testing.

Antibodies doi: 10.3390/antib15010014

Authors: Qiqi Li Zhengyuan Huang Zainab Saeed Orene Greer James A. Harker Nishel M. Shah

The transplacental transfer of maternal immunoglobulin G from the mother to the foetus is central for providing immunity in early life, resulting in full-term newborns having IgG repertoires and levels similar to those of their mothers. The neonatal Fc receptor is recognised as the primary transporter of IgGs across the placental epithelium. Understanding the mechanisms of transplacental antibody transfer and factors that affect them is essential in optimising maternal vaccination strategies, ultimately protecting infants from various environmental pathogens. This review first outlines the biological mechanisms governing transplacental IgG transfer, followed by a discussion of how this process may be disrupted by physiological and pathological conditions during pregnancy, including preterm birth, hypergammaglobulinemia, maternal pathogenic IgG, maternal infections, hyperglycaemia, and exposure to biological therapies. We also summarise currently available models used to study transplacental IgG transfer, highlighting existing knowledge gaps and future directions for research in this field.

Antibodies doi: 10.3390/antib15010013

Authors: Juan González-Fernández Laura Ullate Marta Rodero Alvaro Daschner Carmen Cuéllar

Background/Objectives: Allergic features of anisakiasis, caused by ingestion of third-stage larvae of Anisakis simplex via raw or undercooked fish, manifest clinically as acute gastroallergic anisakiasis (GAA) or chronic urticaria with Anisakis sensitization (CU+). Differentiating these clinical phenotypes remains challenging. This study aimed to evaluate the maturation and avidity of specific antibodies (IgE, IgG4, IgG, and IgA) as biomarkers for discriminating between acute and chronic forms of anisakiasis. Methods: A prospective cohort of 65 patients from Madrid, Spain, was classified into three groups: GAA (n = 22), CU+ (n = 22), and chronic urticaria without sensitization (CU−, n = 21). Serum samples were analyzed for antigen-specific immunoglobulins using ELISA and Western blot. Avidity indices (AIs) were quantified through urea dissociation assays. Statistical comparisons and correlation analyses were performed to associate antibody avidity with clinical phenotype and demographic variables. Results: GAA patients exhibited significantly lower IgE avidity indices compared to CU+ individuals (mean AI: 79.9% vs. 88.5%), indicating a less mature IgE response during acute infection. Conversely, IgG4 and IgG avidity were elevated in GAA relative to CU+, reflecting an active but transient immune response. IgA antibodies were detected in both groups, although avidity differences lacked discriminatory capacity. No sex- or age-related differences in antibody avidity were observed. Longitudinal follow-up of GAA patients demonstrated an increase in IgE avidity over time. Conclusions: Quantitative assessment of antibody avidity, particularly for IgE and IgG4, enhances understanding of A. simplex immunopathogenesis and serves as a valuable biomarker for distinguishing acute from chronic clinical presentations. These findings support the use of avidity indices in the diagnosis, staging, and clinical management of anisakiasis.

Antibodies doi: 10.3390/antib15010012

Authors: Anne-Sophie Kuhlmann Tami Peters Donald K. Hamlin Yawen Li Xinyi Wang Megan Stackhouse Frances M. Cole Jasmin Martinez-Reyes Brenda M. Sandmaier Hans-Peter Kiem D. Scott Wilbur Robert D. Harrington Seth H. Pincus

Background: We are developing cytotoxic immunoconjugates (CICs) to eliminate HIV-infected cells. We investigated the efficacy and kinetics of killing by different forms of CICs targeted by the same monoclonal antibody (mAb), an immunotoxin (IT), antibody-drug conjugate (ADC), and radioimmunoconjugate (RIC). Methods: We compared in vitro effects of CICs made by conjugating anti-gp41 mAb 7B2 to deglycosylated ricin A chain (7B2-dgA), the anthracycline derivative PNU-159682 (7B2-PNU), or the α-emitting isotope actinium-225 (7B2-225Ac). Kinetic analyses of cell growth were performed measuring electrical impedance every 15 min over a 7-day period using cells stably expressing the HIV envelope and Env-negative parent cells. Results: 7B2-dgA and 7B2-225Ac were more potent and acted more rapidly to kill cells than 7B2-PNU. Both the 7B2-PNU and 7B2-225Ac induced bystander-cell killing, whereas the IT did not and consequently allowed the outgrowth of Env-negative cells. Low dose or brief exposure to 7B2-PNU resulted in an increased rate of cell growth. Conclusions: An IT, ADC, and RIC showed substantial differences in the degree of specific toxicity, kinetics, and mechanisms of killing. The results of this side-by-side comparison have implications for the development of CICs to treat HIV, as well as other conditions.

Antibodies doi: 10.3390/antib15010011

Authors: Jilei Wu Chenghua Liu Fenghao Peng Zeyuan Yu Chunxia Qiao Guang Yang Heng Luo Keyi Sun Ziyao Ning Jing Wang Yan Wen Jijun Yu

Background: Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli (EHEC), is a highly potent exotoxin responsible for severe complications such as hemolytic uremic syndrome (HUS). Among its isoforms, Stx2 exhibits stronger cytotoxicity and poses greater clinical risk, yet no effective therapy currently exists. Methods: In this study, two human monoclonal antibodies, YG12-1 and YG12-2, were identified from a phage display library and systematically characterized using an integrated modeling–validation workflow. Results: Structural modeling with ImmuneBuilder and Rosetta revealed that YG12-2 possessed a longer CDRH3 topology, more short-range hydrogen bonds, and stronger electrostatic complementarity, corresponding to lower binding energy and higher apparent affinity in ELISA and SPR. Although YG12-2 had a better affinity, YG12-1 shows better protective activity in a murine model of acute peritoneal infection. This paradox highlights a non-linear relationship between structural affinity and biological efficacy, emphasizing the importance of functional epitope accessibility and pharmacokinetic behavior in determining neutralization outcomes. Conclusions: Overall, these results indicated that targeting Stx2 with YG12-1 and YG12-2 could serve as a promising protective strategy against E. coli O157:H7 infection.

Antibodies doi: 10.3390/antib15010010

Authors: Yukihiro Shoyama

There are a vast number of monoclonal antibodies (MAbs) against biological components; however, the number for natural products is less than 50. MAbs against ginsenosides, i.e., dammarane triterpene glycosides contained in ginseng, were prepared to develop an Eastern blotting method that can estimate the number of bound sugars and pharmacological activity. Meanwhile, as a method for producing ginsenoside Rg3, which is used as an anti-cancer drug, an affinity column for ginsenoside Rb1 was prepared to isolate the raw material ginsenoside Rb1 in a single step, and a method for obtaining ginsenoside Rg3 through fermentation was proposed. A unique MAb capable of detecting all solasodine glycosides contained in Solanum plants was created to prepare an affinity column capable of isolating solasodine glycosides from S. khasianum fruit in a single step. The single-chain variable fragment gene was induced from the MAb against solasodine glycoside and introduced into the hairy root system of S. khasianum, thereby increasing the solasodine glycoside content more than twofold. As a result, we recognized that this method can be used to breed plants with higher concentrations of plant secondary metabolites like solasodine glycosides. The above results collectively demonstrate that solasodine glycoside can be isolated from S. khasianum in high yields and that this compound enables the production of steroids in high yields through a one-step chemical reaction.

Antibodies doi: 10.3390/antib15010009

Authors: Ming-Ching Hsieh Chao Richard Li Margaret A. Velardo Jingming Zhang Babita S. Parekh

Background: This study assesses the robustness of a legacy N-glycan profiling method for the therapeutic antibody MAB1 with different Peptide-N-glycosidase F (PNGase F) enzyme sources, solid phase extraction (SPE) cartridges, and reagent stability, aligning with ICH Q14 lifecycle management principles. Glycosylation profiling is critical for therapeutic antibodies as it influences both function and pharmacokinetics. Method: The legacy N-glycan profiling method, 2-aminobenzoic acid (2-AA) labeling combined with normal-phase HPLC, was re-evaluated to confirm consistent analytical performance in the context of evolving regulatory expectations. The evaluation focused on three key factors: PNGase F enzyme sources, solid-phase extraction (SPE) cartridges, and reagent stability. Results: Commercial PNGase F enzymes showed various performances, with some sources yielding significant differences. Several SPE cartridges were also tested, with certain formats displaying poor recovery and high variability, particularly for sialylated glycans. In addition, reagent stability studies revealed rapid degradation of the labeling reagent within a few days. Conclusions: These results underscore the importance of risk control, continual improvement, and lifecycle management to ensure reliable glycosylation analysis and the sustained robustness of legacy methods.

Antibodies doi: 10.3390/antib15010008

Authors: Stoimen Dimitrov Mihael Tsalta-Mladenov Plamena Kabakchieva Tsvetoslav Georgiev Silva Andonova

Paraneoplastic neurological syndromes (PNSs) are immune-mediated disorders caused by an antitumor response that cross-reacts with the nervous system, leading to severe and often irreversible neurological disability. Once considered exceedingly rare, PNSs are now increasingly recognized owing to the identification of novel neural autoantibodies, wider use of commercial testing, and the emergence of immune checkpoint inhibitor (ICI)-related neurotoxicity that phenotypically overlaps with classic PNS. In this narrative review, we performed a structured search of PubMed/MEDLINE, Scopus, Web of Science, and Google Scholar, without date restrictions, to summarize contemporary advances in the epidemiology, pathogenesis, diagnosis, and management of PNS. Population-based data show rising incidence, largely reflecting improved ascertainment and expanding indications for ICIs. Pathogenetically, we distinguish T-cell-mediated syndromes associated with intracellular antigens from antibody-mediated disorders targeting neuronal surface proteins, integrating emerging concepts of molecular mimicry, tumor genetics, and HLA-linked susceptibility. The 2021 PNS-Care criteria are also reviewed, which replace earlier “classical/non-classical” definitions with risk-stratified phenotypes and antibodies, and demonstrate superior diagnostic performance while underscoring that “probable” and “definite” PNS should be managed with equal urgency. Newly described antibodies and methodological innovations such as PhIP-Seq, neurofilament light chain, and liquid biopsy are highlighted, which refine tumor search strategies and longitudinal monitoring. Management principles emphasize early tumor control, prompt immunotherapy, and a growing repertoire of targeted agents, alongside specific considerations for ICI-associated neurological syndromes. Remaining challenges include diagnostic delays, limited high-level evidence, and the paucity of validated biomarkers of disease activity. Future work should prioritize prospective, biomarker-driven trials and multidisciplinary pathways to shorten time to diagnosis and improve long-term outcomes in patients with PNS.

Antibodies doi: 10.3390/antib15010007

Authors: Muhammad Soyfoo Julie Sarrand

Autoantibodies have long been regarded as passive reflections of immune dysregulation in connective tissue diseases (CTDs). Recent advances in systems immunology and molecular pathology have fundamentally redefined them as active molecular fingerprints that delineate distinct disease endophenotypes with predictive power for clinical trajectories and therapeutic responses. Rather than mere epiphenomena, autoantibodies encode precise information about dominant immune pathways, organ tropism, and pathogenic mechanisms. This review synthesizes emerging evidence that autoantibody repertoires—defined by specificity, structural properties, and functional characteristics—stratify patients beyond traditional clinical taxonomy into discrete pathobiological subsets. Specific signatures such as anti-MDA5 in rapidly progressive interstitial lung disease, anti-RNA polymerase III in scleroderma renal crisis, and anti-Ro52/TRIM21 in systemic overlap syndromes illustrate how serological profiles predict outcomes with remarkable precision. Mechanistically, autoantibody pathogenicity is modulated by immunoglobulin isotype distribution, Fc glycosylation patterns, and tissue-specific receptor expression—variables that determine whether an antibody functions as a biomarker or pathogenic effector. The structural heterogeneity of autoantibodies, shaped by cytokine microenvironments and B-cell subset imprinting, creates a dynamic continuum between pro-inflammatory and regulatory states. The integration of serological, transcriptomic, and imaging data establishes a precision medicine framework: autoantibodies function simultaneously as disease classifiers and therapeutic guides. This endophenotype-driven approach is already influencing trial design and patient stratification in systemic lupus erythematosus, systemic sclerosis, and inflammatory myopathies, and is reshaping both clinical practice and scientific taxonomy in CTDs. Recognizing autoantibodies as endophenotypic determinants aligns disease classification with pathogenic mechanism and supports the transition towards immunologically informed therapeutic strategies.

Antibodies doi: 10.3390/antib15010006

Authors: Giovanni Lasagni Laura Vetrugno Chiara Maria Maggiore Chiara Galassetti Giulia Di Colo Francesco Pavan Andrea Costantino Lorenzo Dagna

Background: Food allergy is a growing public health concern, and oral immunotherapy (OIT) has emerged as a promising approach to induce desensitization and potentially sustained unresponsiveness to allergenic foods. Changes in humoral immunity, particularly in allergen-specific immunoglobulin levels, play a central role in the immunological mechanisms underlying OIT. This review aims to summarize the current evidence on how OIT modulates allergen-specific immunoglobulin E (IgE), G (IgG) and A (IgA) responses in individuals with food allergy. Methods: We conducted a review of original research articles reporting longitudinal data on allergen-specific IgE, IgG, and/or IgA in patients undergoing OIT for common food allergens. Results: OIT was consistently associated with a transient increase in allergen-specific IgE levels during early phases, followed by a gradual decline. In contrast, Allergen-specific IgG4 levels showed a robust and sustained increase, correlating with desensitization and proposed to function as blocking antibodies. Several studies also reported an increase in allergen-specific IgA, particularly secretory IgA at mucosal sites, suggesting a potential role in enhancing mucosal tolerance and immune exclusion of allergens. Conclusions: Humoral immune responses during OIT are characterized by distinct and dynamic changes in immunoglobulin patterns. In particular, the rise in IgG4 and, in some cases, IgA suggests a role in promoting tolerance. Monitoring these biomarkers may offer insights into treatment efficacy and support individualized approaches to OIT.

Antibodies doi: 10.3390/antib15010005

Authors: Hannah Victoria Giles Bhuvan Kishore

Bispecific antibodies (BsAbs) have emerged as an important new class drugs for the treatment of multiple myeloma (MM) over the last few years. Currently, BsAbs are only licensed for use as monotherapy in patients with relapsed/refractory MM who have had at least three prior lines of treatment and are triple class-exposed (patients who have received an anti-CD38 monoclonal antibody, an immunodulatory drug, and a proteasome inhibitor). However, their use in earlier lines, including in the upfront setting, is being explored in multiple ongoing clinical trials with promising early results. The BsAbs have specific toxicities, including a high rate of low-grade cytokine release syndrome and, less commonly, immune effector cell-associated neurotoxicity syndrome. These immune-related toxicities occur almost exclusively during the initiation phase of the BsAbs. This has led to frequent hospitalization of patients for the duration of the initial step-up dosing phase. Strategies that could facilitate outpatient step-up dosing, such as tocilizumab prophylaxis, will become even more critical if BsAbs move into earlier lines of treatment and are used in larger numbers of patients. Optimizing infection prophylaxis is critical for ensuring the safe delivery of BsAbs as infection is the leading cause of non-relapse mortality in patients being treated with BsAbs. Multiple strategies to minimize the infection risk, including antimicrobial prophylaxis, immunoglobulin replacement, vaccination and reduced dosing frequency, have been evaluated. The clinical data on the efficacy of these supportive measures are described in this review article alongside the available strategies for mitigating and managing CRS and ICANS.

Antibodies doi: 10.3390/antib15010004

Authors: Kinsley Wang Kyle Taing Robert Hsu

Background/Objectives: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid proliferation, early metastasis, and near-universal relapse after initial therapy. While chemo-immunotherapy modestly improves first-line outcomes, survival after progression remains poor and highlights the urgent need for biomarker-directed strategies. Methods: A comprehensive literature search was conducted using major medical databases looking at key relevant studies on SCLC antibody studies. All authors reviewed the literature, assessed study quality, and interpreted the results from each study. Results: Recent advances in antibody–drug conjugates (ADCs) and T-cell engagers (TCEs) have transformed therapeutic development by targeting antigens selectively expressed on SCLC cells, enabling more precise and potentially durable tumor control. DLL3 has emerged as the most clinically relevant target to date, with the bispecific TCE tarlatamab demonstrating meaningful and durable response, manageable cytokine-release toxicity, and ultimately achieving accelerated FDA approval for previously treated extensive-stage SCLC. Concurrently, DLL3-directed ADCs have shown variable efficacy, underscoring the importance of payload selection, linker chemistry, and antigen density. Beyond DLL3, next-generation ADCs targeting TROP2, B7-H3, and SEZ6 have reported encouraging early-phase activity, including response rates exceeding those of existing second-line cytotoxic options, though myelosuppression, interstitial lung disease, and hepatic toxicity remain key considerations. Conclusions: Collectively, these emerging immunotherapies illustrate a shift toward antigen-specific targeting in a disease historically defined by limited therapeutic innovation. Continued optimization of antigen selection, payload and linker engineering, and biomarker-driven trial design will be critical for translating early promise into durable clinical benefit and reshaping the treatment landscape for SCLC.

Antibodies doi: 10.3390/antib15010003

Authors: Anna Balata Katarzyna Pogoda

Breast cancer remains the most common malignancy and one of the leading causes of cancer-related death among women worldwide. Advances in antibody-based therapies have improved outcomes across all biological subtypes: HER2-positive, triple-negative, and luminal breast cancer. Monoclonal antibodies such as trastuzumab and pertuzumab have established HER2-targeted therapy as a standard of care, while immune checkpoint inhibitors have introduced immunotherapy into the treatment of triple-negative breast cancer. The emergence of antibody–drug conjugates (ADCs), including trastuzumab deruxtecan, sacituzumab govitecan, and datopotamab deruxtecan, has further expanded the available therapeutic options. Bispecific antibodies represent a new generation of agents with the potential to overcome resistance and enhance immune activation. Despite impressive progress, important challenges remain, including resistance mechanisms and the management of treatment-related toxicities. This review summarizes the biological rationale, clinical evidence, resistance mechanisms, and safety profiles of therapies based on monoclonal antibodies, bispecific antibodies, and antibody–drug conjugates in breast cancer. The development of these treatment modalities fosters the implementation of personalized, immunologically informed treatment strategies that are redefining precision oncology in breast cancer.

Antibodies doi: 10.3390/antib15010002

Authors: Alexandra Rak Yana Zabrodskaya Pei-Fong Wong Irina Isakova-Sivak

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections.

Antibodies doi: 10.3390/antib15010001

Authors: Deepika Godugu Kranthi Gattu Parul Suri Abel B. Daartey Krishna Jadhav Satish Rojekar

Nanobodies (single-domain antibodies, VHHs) have emerged as versatile tools for evaluating and treating Alzheimer’s disease (AD). They offer distinct engineering benefits compared with traditional antibodies and small molecules, including small size, stability, and specificity. In AD, nanobodies have been shown in preclinical models to neutralize toxic amyloid-β oligomers, inhibit tau generation and aggregation, and modulate neuroinflammation, thereby demonstrating significant therapeutic potential. However, all nanobody applications in AD are discussed strictly as preclinical therapeutic potential rather than established clinical therapies, and direct clinical evidence in patients with AD is still lacking. Advanced engineering strategies, including intranasal and intrathecal routes, receptor-mediated transport, plasma protein binding with albumin, and focused ultrasound to facilitate brain penetration. Additionally, to improve nanobody delivery precision, half-life, and efficacy, strategies such as integrating nanobodies with nanoparticles, dendrimers, liposomes, and viral vectors are being employed. In fact, nanobodies are applied beyond monotherapy across multiple technological platforms to optimize brain delivery and target multiple targets. Nanobodies have been used on bispecific and trispecific antibody platforms, as well as in CRISPR/Cas9 editing and AI-driven technologies, to expand their applications. Recently, preclinical evidence has been mounting on the efficacy of nanobodies in clearing Aβ and tau, preserving synapses, and normalizing biomarkers. Comparison with FDA-approved anti-Aβ monoclonal antibodies (aducanumab, lecanemab, and donanemab) highlights opportunities and current translational gaps, including safety testing, half-life extension, and delivery optimization. This review critically delineates the current molecular mechanisms, emerging strategies, and delivery platforms, and emphasizes the potential of nanobodies as promising therapeutic and diagnostic molecules in AD therapeutics.

Antibodies doi: 10.3390/antib14040108

Authors: Jung Gu Lee Inseo Lee Joo-young Kim Suin Kim Woo-jin Jeong Ji-eun Kim

Background: The Fc region of immunoglobulin G (IgG) is a key target in therapeutic and analytical applications, such as antibody purification and site-specific bioconjugation. Although Protein A exhibits strong Fc-binding affinity, its large molecular weight and limited chemical flexibility pose challenges for use in compact or chemically defined systems. To address these limitations, we designed two α-helical peptides, SpA h1 and SpA h2, based on the Fc-binding helices of the Z34C domain from Staphylococcus aureus Protein A. Method: To enhance the structural stability and Fc-binding capability of these peptides, a lactam-based stapling strategy was employed by introducing lysine and glutamic acid residues at positions i and i + 4. Result: The resulting stapled peptides, (s)SpA h1 and (s)SpA h2, exhibited significantly improved α-helical content and IgG-binding performance, as demonstrated by circular dichroism (CD) spectroscopy and fluorescence-based IgG capture assays. Surface plasmon resonance (SPR) analysis confirmed specific, concentration-dependent interactions with the Fc region of human IgG, with (s)SpA h1 consistently showing the binding affinity and stability. Proteolytic resistance assays using α-chymotrypsin revealed that (s)SpA h1 maintained its structural integrity over time, exhibiting markedly enhanced resistance to enzymatic degradation compared to its linear counterpart. Furthermore, (s)SpA h1 exhibited strong Fc selectivity with minimal Fab affinity, confirming its suitability as a compact and Fc-specific binding ligand. Conclusions: These results confirm the successful design and development of structurally reinforced Fc-binding peptides that overcome the inherent limitations of short linear sequences through both high-affinity sequence optimization and lactam-based stapling. Among them, (s)SpA h1 demonstrates the most promising characteristics as a compact yet stable Fc-binding ligand, suitable for applications such as antibody purification and site-specific bioconjugation.

Antibodies doi: 10.3390/antib14040107

Authors: Barbara Colitti Consiglia Longobardi Gabriela Flores-Ramirez Chiara Nogarol Ludovit Skultety Gianmarco Ferrara

Background/Objectives: The detection of anti-Coxiella antibodies using serological methods is essential for identifying exposed ruminants and preventing this important zoonotic disease in livestock. In recent years, numerous attempts have been made to increase diagnostic performance as well as simplify the production of serological assays. Commercially available tests often use whole-cell antigens, which can decrease specificity and require high-level biosafety facilities for manufacturing. The aim of this work was to produce three Coxiella burnetii (C. burnetii) antigens in recombinant form and assess them for the detection of anti-Coxiella antibodies in ruminants. Methods: Three recombinant C. burnetii antigens (Com-1, MceB, AdaA) were selected among immunodominant antigens and produced in a heterologous system (Escherichia coli). Following purification, the proteins were utilized to coat ELISA plates and evaluated for seroreactivity against sera from both negative and positive cattle. Results: Com-1 demonstrated the greatest agreement with the commercial test, albeit moderate. MceB exhibited nonspecific reactivity against a large number of sera, while the AdaA showed reactivity against only a few positive sera. Conclusions: Our findings are consistent with previous research, indicating that utilizing a single antigen to identify exposed animals is unfeasible with current knowledge, most likely due to the complex immunological response following C. burnetii infection in cattle. Consequently, it is critical to continue testing and identifying immunoreactive antigens in order to further investigate them and, potentially, select the most appropriate.

Antibodies doi: 10.3390/antib14040106

Authors: Chia-Hung Chen Yu-Chi Chiu Kai-Yao Huang Hsiao-Hsuan Huang Ta-Wei Kuo Yu-Chi Liu Hui-Ju Kao Chen-Lin Yu Shun-Long Weng Kuang-Wen Liao

Background: Short peptide epitopes are valuable for mechanistic studies, yet their intrinsic low immunogenicity and lack of commercial antibodies hinder rapid antibody generation. Methods: We developed a reproducible, sequence-level workflow combining cross-species/structural triage, independent MHC-I/II prioritization, and conservative heteroclitic-style substitutions to enhance predicted MHC affinity while preserving native epitope features. Using visfatin as a model, two optimized fragments were conjugated to KLH and tested in mice for antibody titers, isotype profiles, and binding kinetics. Results: Mutant peptides improved MHC-binding prediction, elicited stronger antibody titers, and promoted isotype maturation (increased IgG1). Importantly, antibodies maintained measurable binding to wild-type sequences, indicating preserved cross-recognition. Similar effects were reproduced with additional antigens. Conclusions: This proof-of-concept study, based on small exploratory mouse cohorts (n = 3 per group), demonstrates that strategic, minimal sequence edits can significantly enhance peptide immunogenicity while preserving native epitope recognition. This streamlined workflow provides a low-barrier route to generate epitope-directed antibodies when commercial reagents are unavailable.

Antibodies doi: 10.3390/antib14040105

Authors: Yuxin Qian Weiwei Ma Xiao-Ning Xu

Chimeric antigen receptor (CAR)-based immunotherapy has emerged as a transformative strategy in anticancer treatment, driven by advances in CAR construct design, manufacturing platforms, and expansion to diverse immune cell types. The landmark success of CD19-targeted CAR-T cell therapy in B cell malignancies has paved the way for broader clinical applications. As of 2025, the U.S. FDA has approved multiple autologous CAR-T products, underscoring their therapeutic promise. However, challenges persist, including cytokine release syndrome (CRS), neurotoxicity, product inconsistency, and the high cost and complexity of cell manufacturing. Variations in cell source, gene delivery methods, expansion protocols, and CAR design significantly influence the safety, efficacy, and scalability of these therapies. In this review, we comprehensively examine the current advances in manufacturing protocols for CAR-modified T cells, natural killer (NK) cells, and unconventional T cell subsets, including γδ T, invariant natural killer T (iNKT), and mucosal-associated invariant T (MAIT) cells. We also highlight emerging innovations such as in vivo CAR-T generation and off-the-shelf allogeneic approaches. By integrating updated strategies with a critical evaluation of current limitations, this review aims to support the development of standardized, robust, and accessible CAR-based immunotherapies.

Antibodies doi: 10.3390/antib14040104

Authors: Giuseppe Lauletta Cataldo Patruno Claudio Brescia Andrea Cosenza Carolina D’Elia Valentina Ventura Emanuela Martina Maddalena Napolitano

Background: Head and neck dermatitis (HND) represents a challenging phenotype of atopic dermatitis (AD), often showing suboptimal response or paradoxical worsening during biologic therapy. Objective: To review the efficacy and safety of current systemic treatments for HND, with a focus on dupilumab, tralokinumab, lebrikizumab, and janus kinase (JAK) inhibitors. Methods: We conducted a narrative review of randomized controlled trials, post hoc analyses, and real-world studies assessing clinical outcomes in patients with moderate-to-severe AD involving the head and neck. Outcomes included Eczema Area and Severity Index (EASI) H&N subscore, erythema grade, patient-reported measures, and adverse events. Results: Dupilumab shows substantial efficacy for HND in both clinical trials and real-life studies; however, responses are often less pronounced than in other anatomical regions, and facial redness (FR) has emerged as a notable adverse event in up to 9% of patients. Tralokinumab and lebrikizumab demonstrate significant improvements in HND involvement, with low incidence of paradoxical reactions. JAK inhibitors, particularly upadacitinib, provide rapid and marked improvement in refractory cases and in patients developing FR during biologic therapy. Conclusions: Systemic therapy for HND should be individualized, balancing efficacy and tolerability. JAK inhibitors represent a valuable alternative in biologic-refractory phenotypes or in patients experiencing dupilumab-associated FR.

Antibodies doi: 10.3390/antib14040103

Authors: Thais Stelzer Toledo Pauline Martins Cunha Josué da Costa Lima-Junior Monique Paiva De Campos Alinne R. S. Renzetti Fabiano Borges Figueiredo Fernanda Nazaré Morgado Renato Porrozzi Fatima da Conceição-Silva Marta de Almeida Santiago Paula Mello De Luca

Backgroud/Objectives: Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern due to dogs serving as reservoirs for human infection. An accurate and rapid diagnostic method to distinguish symptomatic and asymptomatic CVL from healthy and vaccinated animals is essential for controlling canine and human disease. Developing innovative antibody detection techniques and exploring new antigens are essential for enhancing CVL testing efficiency. Our study focuses on a multiplex flow cytometry technique to detect Leishmania-specific antibodies in canine serum. This involved conjugating small peptides with carrier proteins or peptide tags, sequences designed to facilitate bead coupling. Methods: A peptide from the L. infantum A2 protein was coupled to beads in three forms: unconjugated, conjugated with BSA, and conjugated with a C-terminal β-alanine–lysine (x4)–cysteine TAG. This TAG was previously designed to enhance peptide solubility, improve binding efficiency, and provide functional groups for covalent attachment to the beads, ensuring stable immobilization in the multiplex assay. Results: Our results suggest that the multiplex approach shows promise as a rapid serological test for CVL, particularly with TAG-conjugated peptides, which optimize bead coupling. However, peptide/BSA conjugation revealed anti-BSA antibodies in samples from healthy and CVL dogs. Conclusions: In conclusion, our findings highlight the potential of multiplex methodologies to enhance CVL diagnostics and caution against using BSA as a bead coupling agent in serological tests for canine samples due to its impact on test specificity and sensitivity.

Antibodies doi: 10.3390/antib14040102

Authors: Eun Seo Choi Sophia Sahota Emily Burnham Yunfeng Ding Eric V. Shusta

Background: Antibodies that cross the blood–brain barrier (BBB) by targeting receptor-mediated transport (RMT) systems can allow efficient drug delivery to the central nervous system (CNS). In order to improve brain uptake of antibodies, their binding properties have been engineered, but it is not always clear what antibody properties dictate BBB transport efficiency. In this study, we therefore developed and employed an in vitro phenotypic screen and a quantitative transcytosis assay in an attempt to identify improved variants of a previously identified BBB transcytosing antibody known as 46.1. Methods: First, a random mutagenic 46.1 antibody phage display library was screened for improved transcytosis through a human induced pluripotent stem cell (iPSC)-derived BBB model. These screens yielded antibody variants that enriched over multiple screening rounds; however, when produced as soluble antibodies, the variants did not display improved in vitro transcytosis over the wild-type (WT) 46.1 antibody. As a second strategy, we performed a targeted histidine point mutation of a solvent-exposed residue in each complementarity-determining region (CDR) and evaluated the in vitro transcytosis capacity of the variants. Results and Conclusions: In this way, we identified a 46.1 variant, R162H, with modestly improved in vitro transcytosis properties. These results show that the iPSC-derived BBB screening insights and evaluation strategies presented here could facilitate the engineering and optimization of lead antibodies for CNS delivery.

Antibodies doi: 10.3390/antib14040101

Authors: Garry M. Walsh

Background: Asthma exhibits marked heterogeneity both clinically and at the molecular phenotypic level, requiring specifically targeted treatments to block the key pathways of the disease. Monoclonal-antibody-based biologics targeted at critical inflammatory pathways of T2 inflammation such as IL-5, IL-5R, IL-4, and IL-13 are increasingly regarded as effective treatments for severe refractory eosinophilic asthma. Methods: This review provides an update on the potential of straightforward and reproducible biomarkers to aid in the selection of the biologic-based therapy most likely to be effective in patients with severe or refractory eosinophilic asthma based on English-language original articles in PubMed or MedLine. Results: Monoclonal-antibody-based biologic therapies have revolutionised severe asthma management, enabling reductions in symptoms that include exacerbations, discontinuation of oral corticosteroids, improved lung function, and enhanced quality of life. Significant clinical effects with anti-IL-5 or -IL-4/13 monoclonal antibodies are more likely to be seen when simple predictive biomarkers such as serum periostin, fractional exhaled nitric oxide (FENO), or blood eosinophil counts are used to aid in the identification of those patients with severe refractory eosinophilic asthma who are most likely to benefit from biologic therapies. Conclusions: Biologic-based therapy aimed at T2 inflammation benefits patients with severe eosinophilic asthma, particularly when guided by biomarkers that do not require direct sampling of the airways to target therapy, who are most likely to benefit from these treatments, with good safety profiles for these therapies.

Antibodies doi: 10.3390/antib14040100

Authors: Marta Baselga Javier Sánchez-Prieto Víctor Manuel Medina Pérez Alberto J. Schuhmacher

Background/Objectives: Single-domain antibodies (sdAbs) are derived from camelid heavy-chain antibodies (HCAb). Their small size, high stability, and ease of production, among other properties, makes them highly valuable in biomedical research and therapeutic development. Several sdAb-based molecules are currently progressing through clinical trials, highlighting their translational relevance. As sdAbs originate from HCAb of Camelidae family, they can originate from multiple species including Vicugna pacos, Lama glama, Camelus dromedarius and Camelus bactrianus. Although several reports and databases analyze the structure of sdAbs, comprehensive evaluations on species-dependent structural differences remain scarce. Methods: We assembled MO-IISA, an open-access curated database of sdAbs with known antigen targets by integrating six public resources (iCAN, INDI, SAbDab-nano, sdAb-DB, PLabDab-nano, NbThermo) under harmonized eligibility criteria. Results: The final dataset comprises 2053 sdAbs derived from llamas (Lama glama, n = 1316); alpacas (Vicugna pacos, n = 325), dromedary camels (Camelus dromedarius, n = 377) and Bactrian camels (Camelus bactrianus, n = 35). We quantified region lengths, amino acid frequency, and conservation/entropy across frameworks (FR1–FR4). The average length of all sdAbs was about 124 ± 8 amino acids, with minor interspecies differences. We observed a consistent enrichment of lysines in FR3 (and secondarily FR2) and cysteines primarily in FR1 and FR3, with non-canonical cysteines more frequent in Bactrian and dromedary sdAbs CDRs. CDR2 and, particularly CDR3, contributed most to inter- and intra-species variability, whereas FRs were highly conserved. Conclusions: Species-neutral framework constraints and species-tuned loop adaptations have practical implications for sdAb engineering, species selection, and conjugation strategies. These features are captured in MO-IISA, an open-access database of known-target sdAbs from different species.

Antibodies doi: 10.3390/antib14040099

Authors: Million A. Tegenge

Background: Physiologically based pharmacokinetic (PBPK) modeling is applied to address clinical pharmacology issues including dose selection and exposure assessments for special populations (e.g., pediatrics, and renally or hepatically impaired patients). The objective of this study was to evaluate the predictive performance of a PBPK model for dosing assessment of intravenous immunoglobulin (IVIG) and anti-D immunoglobulin (anti-D Ig) products in pregnant women. Methods: A minimal PBPK (mPBPK) model that incorporates pregnancy-specific physiological parameters and allometric scaling approaches was developed and evaluated for predicting the exposure of IVIG and anti-D Ig in pregnant women. The concentration versus time data were obtained from the published literature. Results: The IVIG (n = 22) and anti-D Ig (n = 29) concentrations were predicted using the mPBPK model with an average fold error of 1.17 and 1.22, respectively. A total of 100% and 95% of IVIG concentrations were predicted within the 0.5–2-fold and 0.5–1.5-fold prediction error ranges, respectively. For anti-D Ig, predictions fell within the 0.5–2-fold and 0.5–1.5-fold ranges for 93% and 76% concentrations, respectively. A mPBPK model-based simulation following administration of 0.5 g/kg IVIG in 100 virtual nonpregnant and pregnant subjects revealed that the maximum plasma concentration (Cmax) was 15% lower and trough concentration (Ctrough) was 8% lower during the third trimester of pregnancy compared to nonpregnant subjects. In contrast, with flat dosing, Cmax and Ctrough were 32% and 26% lower in pregnant subjects, respectively. Overall, the model demonstrated reasonable predictive performance, and bodyweight-based dosing regimen is an acceptable approach that results in minimal change in exposure of IVIG in pregnant women.

Antibodies doi: 10.3390/antib14040098

Authors: Paolo Giannoni Gabriella Pietra Orlando Izzo Giuseppina Fugazza Roberto Benelli Alessandro Poggi Mauro Krampera Chiara Utzeri Monica Marchese Marco Musso Paola Visconti Daniela de Totero

Background/Objectives: Three-dimensional (3D) in vitro cell culture models have recently stimulated great interest since they may have more pre-clinical value than conventional in vitro 2D models. In fact, 3D culture models may mimic the in vivo biophysical 3D structure of tumors and cell-to-cell interaction, thereby representing a more useful approach to testing drug responses. In this study we have developed a 3D culture model of an EBV+/CD30+cell line, D430B, previously characterized as an Anaplastic Large Cell Lymphoma of B phenotype (B-ALCL), to determine the cytotoxic activity of the antibody–drug conjugate Brentuximab Vedotin. Methods: By using of ultra-low attachment plates, we developed D430B spheroids that appeared particularly homogenous in terms of growth and size. Results: Brentuximab Vedotin treatment (1 to 20 μg/mL) turned out to be significantly cytotoxic to these cells, while the addition of the anti-CD20 chimeric antibody Rituximab (10 μg/mL) appeared almost ineffective, even though these cells express CD20. Moreover, when we co-cultured D430B cells with stromal cells (HS5), to re-create a microenvironment representative of neoplastic cell/mesenchymal cell interactions within the lymph node, we observed a significant, although faint, protective effect. Conclusions: This simple and reproducible method of generating D430B-ALCL spheroids to evaluate their response to Brentuximab Vedotin treatment, as here described, may provide a valuable preliminary tool for the future pre-clinical screening of patients’ primary lymphoma cells or the development of novel therapies for this type of pathology and related diseases.

Antibodies doi: 10.3390/antib14040097

Authors: Saravana P. Selvanathan Olivia O. Lansinger David V. Allegakoen Emma J. W. McGuire Ashley R. Gaffey Jeff R. Petro Purushottam B. Tiwari Quinn Tufiño Aykut Üren Jeffrey A. Toretsky

Background: Ewing sarcoma (ES) is a rare tumor that affects children, adolescents, and young adults. ES is associated with high morbidity in all patients and high mortality for those who present with metastatic disease. A chromosomal translocation, either t(11;22)(q24;p12) or t(21;22)(q22;q12) leads to the fusion oncoproteins EWS::FLI1 or EWS::ERG in 95% of ES patients. We recognized a critical need for a stably sourced high-affinity antibody that recognizes EWS::FLI1 with maximal specificity. Understanding EWS::FLI1 protein complexes is a pivotal gap in ES knowledge that necessitates the development of antibodies capable of identifying native proteins in solution. Further, variable epitope sequencing of a monoclonal antibody enables the construction of degraders and nanobody identifiers. Methods: Monoclonal antibodies were produced following informed peptide synthesis, injection, and hybridoma creation. Hybridoma antibodies were validated for specificity and function. Results: Our results indicate that the FLI1 1.2 monoclonal antibody, which recognizes the EWS::FLI1 fusion oncoprotein, can be reliably applied to multiple molecular biology applications like immunoblot, immunoprecipitation, immunofluorescence, and immunohistochemistry. This FLI1 1.2 monoclonal antibody has a high affinity of 0.3 nM KD to EWS::FLI1. In terms of specificity, this antibody is highly specific to EWS::FLI1 and some cross reactivity with ERG. Conclusions: This reagent will provide the research community with valuable tools for further biochemical and genomic interrogation of the oncogenic activity of EWS::FLI1 in ES.